Method for the simultaneous detection of multiple nucleic acid sequences in a sample

ABSTRACT

Detecting nucleic acid sequences in a sample, such as the detection of pathogenic organisms in clinical samples. More specifically, detecting an infection caused by a pathogenic organism such as a virus or a bacterium in a clinical specimen by means of amplifying and detecting specific nucleic acid sequences from said pathogenic organism. A multiplex assay with the possibility to determine about 30 different target nucleic acid sequences in a single one-tube assay combined with real-time probe detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is filed under 35 U.S.C. §111 as acontinuation-in-part of U.S. application Ser. No. 12/668,067, filed onJan. 7, 2010, which is a National Stage Entry of InternationalApplication No. PCT/EP2008/059050, filed on Jul. 10, 2008, whichdesignates the United States and claims the benefit of EuropeanApplication No. 07112219.6, filed on Jul. 11, 2007, the contents ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the technical field of detecting multiplenucleic acid sequences in a sample, such as the detection of pathogenicorganisms in clinical samples. More specifically, the invention relatesto the field of detecting an infection caused by a pathogenic organismsuch as a virus or a bacterium in a clinical specimen by means ofamplifying and detecting specific nucleic acid sequences from saidpathogenic organism.

BACKGROUND OF THE INVENTION

Infectious agents such as micro-organisms are typically detected byculturing clinical samples under conditions favourable for the growth ofsuch micro-organisms and monitoring that growth by a number of differenttechniques including microscopy and detection of more or less specificmetabolites of the organisms.

Nucleic acid amplification tests to identify pathogens rapidly andreliably have been implemented in the microbiology laboratory during thelast decade. Nucleic acid amplification tests can be used to detect thepresence of micro-organisms directly in clinical specimens withoutculturing.

Initially, identification was accomplished by amplification of a targetnucleic acid sequence and detecting of the resulting DNA byvisualisation using gel electrophoresis and DNA-binding fluorescentdyes.

Nucleic acid amplification tests revolutionized the world of clinicaldiagnosis in that they provided an increase in sensitivity and speed ofan order of several magnitudes as compared to the classical cultureassays.

In general, nucleic acid amplification tests consist of a targetspecific nucleic acid amplification step and a more or less genericdetection step. Herein below follows a brief summary of availableamplification techniques and detection platforms.

PCR is currently still the first choice to amplify target sequences andthe ability to amplify a wide range of pathogens is dependent ongeneric, random or multiplex amplification technologies.

In generic PCR tests, only one or two primers pairs are necessary toamplify a target sequence from a range of related pathogens. Regions ofconserved nucleotide sequences are required and in general degenerateprimer pairs are used.

Several random amplification technologies exist, making use of eitherrandom octamers or primers that contain a random 5-8 nucleotideextension at its 3′-end and a defined sequence at its 5′-end. Randomamplification is performed in combination with Taq polymerase orisothermal polymerase-based amplification enzymes such as Klenow DNApolymerase or Φ29 DNA polymerase.

Multiplex PCR involves the combination of several primers pairstargeting different sequences in one amplification reaction. MultiplexPCRs require careful optimization to make them comparably sensitive andspecific as single pair amplification reactions.

Multiplex Ligation-dependent Probe Amplification (MLPA) technology(Schouten et al; WO 01/61033, Schouten et al., Nucl. Acids Res. 2002,vol 30 No 12; e57) is a multiplex PCR method capable of amplifyingdifferent targets simultaneously. In MLPA two oligonucleotides thathybridise immediately adjacent to each other on target DNA are added inthe same reaction. One of the oligonucleotides is synthetic and has asize of 40-60 nucleotides (nt) whereas the other oligonucleotide has asize ranging from 100 up to 400 nt and requires a cloning step in an M13vector to finally generate single stranded probe DNA. MLPA consists ofthree steps: first an annealing step to hybridise the probes to theirtarget region, secondly a ligation step to covalently link the twoprobes together and thirdly the final PCR to get an exponentialamplification of the target regions using only two universal primers.

Currently, target specific multiplex PCR amplification is the standardmethod for pathogen detection assays.

Because of the extreme sensitivity of nucleic acid amplification tests,care must be taken to avoid contamination in these tests. Detection ofamplified nucleic acids was originally performed by size determinationusing gel electrophoresis and intercalating DNA dyes. A first step inminimizing contamination was taken when the amplification and detectionsteps were combined into one step. As a result, post-amplificationhandling steps were eliminated, thereby adding to the reliability of theassay. Such assays are often referred to as a closed system. Closedsystem amplification technologies such as real time PCR (Ratcliff et al.Curr Issues Mol Biol. 2007, 9(2): 87-102) and NASBA (Loens et al., JClin Microbiol. 2006, 44(4); 1241-1244) and LAMP (Saito R et al., J MedMicrobiol. 2005, 54; 1037-41) have been developed and use intercalatingfluorescent dyes or fluorescent labelled probes. The isothermal LAMPtechnology allows real time detection by spectrophotometric analysisusing a real-time turbidimeter. Currently, most of these assays areorganism-specific and useful only when a particular pathogen issuspected. This limits the scope of these assays considerably.

However, clinical symptoms are only rarely attributable to a singlepathogen. Hence, there is a need in the art for assays that allow thesimultaneous identification and differentiation of multiple agents. Suchmulti-parameter assays enable the clinician to come to a faster andbetter therapy and contribute to improved clinical management and publichealth.

For this reason technologies have been developed for the purpose oftesting simultaneously for more than one organism. One of suchtechnologies is multiplex real time PCR. At present, however, only fivecolour oligo-probe multiplexing is possible of which one colour isideally set aside for an internal control to monitor inhibition andperhaps even acts as a co-amplified competitor (Molenkamp et al. J.Virol Methods, 2007, 141: 205-211). This considerably limits the amountof pathogens that may be tested simultaneously.

An example of an area where it is particularly desirable to have aquick, reliable and specific multiplex assay for several pathogens atonce, is the area of respiratory tract infections.

Acute respiratory tract infection is the most widespread type of acuteinfection in adults and children. The number of pathogens involved isnumerous. Respiratory tract infections (RTI) are commonly divided intoupper respiratory tract infections (URTI) and lower respiratory tractinfections (LRTI). The URTI include rhinorrhea, conjunctivitis,pharyngitis, otis media and sinusitis and LRTI include pneumoniae,brochiolitis and bronchitis. Both viruses and bacteria cause acute RTI,and the number of causative agents is large as well as diverse.

Non-typical viruses and bacteria involved in RTI include influenza virusA and B (InfA and B), parainfluenza virus 1, 2, 3 and 4 (PIV-1, -2, -3and 4), respiratory syncytial virus A and B (RSVA and B), rhinovirus,coronavirus 229E, OC43 and NL63 (Cor-229E, -OC43 and NL63), severe acuterespiratory syndrome coronavirus (SARS-CoV), human metapneumovirus(hMPV), adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae,Legionella pneumophila and Bordetella pertussis. Many of theseinfections are indistinguishable by clinical features alone and requirerapid laboratory tests for optimal patient management and infectioncontrol.

Viral culture is still the gold standard for laboratory diagnosis ofrespiratory viruses. However, viral culture is relatively slow andtherefore routine diagnosis is sub optimal. Although rapid antigendetection tests are available for some of these viruses, these testshave shown to be less sensitive and less specific than viral culturing.Currently, there is a desperate need for a sensitive and specific methodfor the simultaneous detection of respiratory viruses in a multiplexformat. It would be very advantageous to be able to detect two or moretargets in a single reaction as this would provide distinct advantagesin clinical diagnostics. It would simplify the assay, increase thethroughput, minimize the consumption of clinical specimen and inparticular multiplex assays would be more cost-effective than monoplexassays.

To differentially detect respiratory viruses in clinical specimens thefollowing detection platforms have been used:

(i) Gel Electrophoresis.

Using agarose gel electrophoresis as detection device, several nestedmultiplex reverse transcriptase (RT)-PCR assays have been developedusing three or four primer pairs. Osiowy et al., (J. Clin. Microbiol.1998, 36; 3149-3154) used five primers pairs that amplified RNA fromrespiratory syncytial virus A and B, parainfluenza virus 1, 2 and 3 andadenovirus types 1 to 7. The PCR products varied in size from 84 up to348 base pairs. Compared to direct immunofluorescence (DIF) assays orindirect immunofluorescence (IIF) assays a sensitivity value of 91% anda specificity value of 87% was obtained for this multiplex RT-PCRapproach.

Coiras et al. (J. Med. Virology, 2004, 72; 484-495) using the sameapproach, were able to simultaneously detect 14 respiratory viruses intwo multiplex RT nested PCR assays. They included coronavirus 229E andOC43, rhinovirus, enterovirus, parainfluenza virus 4 and an internalcontrol but omitted adenovirus 1 to 7. The assay was evaluated on noseand throat swaps and nasopharyngeal aspirates from infants below twoyears of age. It appeared that the multiplex assay was more sensitivethan conventional viral culture and immunofluorescence assays, with theadvantage that all viruses can be tested at the same time and with asingle technique. In addition, in 9.5% of the samples a double infectionwas found.

Erdman et al. (J. Clin. Microbiol. 2003, 41; 4298-4303) recentlydeveloped a RT-PCR assay against 6 common respiratory viruses based onautomated fluorescent capillary electrophoresis and Genescan softwarefor detection of respiratory syncytial virus A and B, parainfluenzavirus 1, 2 and 3 and influenza virus A and B. An one-step RT-PCRreaction was performed using primers of which the positive strand primerof each primer set was 5′ end labelled with the fluorescent dye6-carboxyfluorescein (6-FAM). Overall, this RT-PCR assay was positive in92% of the samples that were also positive by culture or DIF staining.

The above references are examples of techniques wherein a large numberof samples is analysed using gel-electrophoresis. Disadvantages of gelelectrophoresis as a detection technique are that it is laborious andtime consuming and therefore rather costly. Furthermore, the risk ofcross contamination is enlarged as each sample has to be opened afterthe PCR for analysis.

(ii) Secondary Enzyme Hybridisation.

In this approach multiplex PCR assays are combined with an enzyme-linkedimmunosorbent assay (ELISA).

Detection of the multiplex PCR products is performed by microwellhybridisation analysis in streptavidin-coated wells of a microtiterplate. Biotinylated capture probes specific for the amplified targetsequences are added and a peroxidase labelled hybridisation reaction isperformed. Subsequently, the optical density is measured by areader/spectrophotometer. Samples are classified as PCR positive ornegative depending on the cut-off optical density value.

Multiplex RT-PCR enzyme hybridisation assays for rapid identification ofseven or nine micro-organisms causing a respiratory tract infection havebeen developed and validated in comparison to the gold standard. Thecommercially available Hexaplex assay (Prodesse, Inc., Milwaukee, Wis.)(Fan et al, Clin. Infect. Dis. 1998, 26; 1397-1402, Kehl et al., J.Clin. Microbiol. 2001, 39; 1696-1701 and Liolios et al., J. Clin.Microbiol. 2001, 39; 2779-2783) is directed against parainfluenza virus1, 2 and 3, respiratory syncytial virus A and B and influenza virus Aand B whereas the nineplex assay contained the same RNA viruses minusparainfluenza 2 and respiratory syncytial virus A and B were combined inone primer pair but including enterovirus, adenovirus and two bacteriaMycoplasma pneumoniae and Chlamydia pneumoniae (Grondahl et al., J.Clin. Microbiol. 1997, 37; 1-7 and Puppe et al., J. Clin. Virol. 2003,30; 165-174). The analytical sensitivity of the Hexaplex assay has beenshown to be 100-140 copies/ml depending on the virus, whereas thenineplex assay was less sensitive compared to culture for respiratorysyncytial virus and parainfluenza virus 1 and more sensitive forparainfluenza virus 3, influenza virus A and B, adenovirus andenterovirus. The analytical sensitivity was measured on serial dilutionsof viral culture supernatants. The sensitivity and specificity of thenineplex and hexaplex on clinical specimens varied between the RNAviruses and was found to be between 86% -100% for sensitivity and80%-100% for specificity. Both assays were compared with monoplex RT-PCRELISA and other monoplex RT-PCR tests and were approximately of the samequality. Although the ELISA based assays allow highly multiplexanalyses, they posses the same disadvantages as the gel electrophoresisbased assays. The assays are laborious and time consuming and thereaction vessel has to be opened after PCR, thereby increasing the riskof cross contamination.

(iii) Measuring Emission Using Different Fluorescent Dyes.

Fluorescence reporter systems such as real time PCR have been introducedin the diagnostic laboratory recently. Real Time PCR combines DNAamplification with detection of the products in a single tube. Detectionis based on changes in fluorescence proportional to the increase inproduct. Real Time PCR capacity to simultaneously detect multipletargets is limited to the number of fluorescent emission peaks that canbe unequivocally resolved. At present, only four colour oligoprobemultiplexing is possible of which one colour is ideally set aside for aninternal control to monitor inhibition and perhaps even acts as aco-amplified competitor.

Many monoplex or duplex real-time PCR assays against respiratorypathogens have been developed either being home brew based (van Elden etal., J. Clin. Microbiol. 2001, 39; 196-200, Hu et al, J. Clin.Microbiol. 2003, 41; 149-154) or commercially available assays(Prodesse, Inc., Milwaukee, Wis.). One of the first multiplex real-timePCR assays directed against respiratory viruses was developed byTempleton et al. (J. Clin. Microbiol. 2004, 42; 1564-1569). A real-timemultiplex PCR assay was developed for the detection of 7 respiratory RNAviruses (influenza virus A and B, respiratory syncytial virus,parainfluenza virus 1, 2, 3 and 4) in a two-tube multiplex reaction.Each assay was initially set up as a monoplex assay and then combined intwo multiplex assays: one comprises influenza virus A and B andrespiratory syncytial virus whereas as the other one comprisesparainfluenza virus 1, 2, 3 and 4 with both assays having the same PCRprotocol so they could be run in parallel. No non-specific reactions orany inter-assay cross-amplification was observed and only the correctvirus was amplified by the two multiplex reactions. Clinical evaluationwas performed by viral culture and confirmed by IF and multiplex PCR onthe same samples. Viral culture resulted in 19% positive samples whereasmultiplex resulted in 24% positives. The multiplex PCR-positivespecimens included all the samples that were positive by viral cultureand additional ones. The additional ones were tested by a secondPCR-assay and it could be shown that that these samples were truepositives.

For simultaneous detection of 12 respiratory RNA viruses by real-timePCR, Gunson et al. (J. Clin. Virol. 2005, 33; 341-344) developed fourtriplex reactions: (i) influenza virus A and B and humanmetapneumovirus, (ii) respiratory syncytial virus A and B andrhinovirus, (iii) parainfluenza virus 1, 2 and 3 and (iv) coronavirus229E, OC43 and NL63. These 4 assays cover almost the complete set ofrespiratory RNA viruses and implementation of these assays was said toimprove patient management, infections control procedures and theeffectiveness of surveillance systems. The real time PCR assays allowanalysis without any post PCR handling of the sample. This diminishesthe risk of cross contamination and requires no extra handling time.However, the complexity of the current assays is limited to a maximum offour probes per reaction. Moreover, complex analyses require morereactions thereby increasing the costs.

(iv) Microarrays Consisting of Oligonucleotides or PCR AmpliconsImmobilized on a Solid Surface.

Microarrays for diagnostic purposes require either (a) genome specificprobes to capture the unknown target sequences or (b) generic zipcodespresent in the amplified target sequence and thereby reveal the presenceof that pathogen in a clinical specimen. Hybridisation between the boundprobe and target sequence in the sample is revealed by scanning orimaging the array surface.

DNA microarrays offer the possibility for highly parallel viralscreening to simultaneously detect hundreds of viruses. Related viralserotypes could be distinguished by the unique pattern of hybridisationgenerated by each virus. High density arrays are able to discriminatebetween ten thousand different targets whereas low density arrays of upto a few hundred targets are more appropriate in clinical diagnostics.The first array for use in diagnostic virology was constructed by Wangat al. (Proc. Natl. Acad. Sci. USA 99; 15678-15692). They initiallyconstructed a microarray of 1600 unique 70-mer oligonucleotide probesdesigned from about 140 viral genome sequences of which the respiratorytract pathogens were of major concern. The viral RNA was amplified usinga randomly labelled PCR procedure and the array was validated with nasallavage specimens from patients with common colds. The array detectedrespiratory pathogens containing as few as 100 infectious particles. Thedata were confirmed with RT-PCR using specific PCR primers. Crosshybridisation was only observed to its close viral relatives.

Low density arrays have been constructed for detection, typing andsub-typing of Influenza (Kessler et al., J. Clin. Microbiol. 2004, 42;2173-2185) and acute respiratory disease-associated adeno viruses (Linet al., J. Clin Microbiol. 2005, 42; 3232-3239). The Influenza chip wasshown to detect as few as 1×10² to 5×10² influenza virus particleswhereas the sensitivity of the adeno microarray was 10³ genomic copieswhen clinical samples were analysed directly. Multiplex as well asrandom amplification procedures were used.

A very new development in respiratory tract pathogen identification isthe use of re-sequencing microarrays (Lin et al., Genome Res., 2006,16:527-535, and Wang et al, Bioinformatics 2006 22(19):2413-2420;doi:10.1093/bioinformatics/bt1396). The exponentially increasingavailability of microbial sequences makes it possible to use directsequencing for routine pathogen diagnostics. However, this requires thatpathogen sequence information be rapidly obtained. Resequencingmicroarrays use tiled sets of 10⁵ to 10⁶ probes of either 25-mers or29-mers, containing one perfectly matched and three mismatched probesper base for both strands of target genes. A custom designed Affymetrixre-sequencing Respiratory Pathogen Microarray (RPM v.1) has beendisclosed. This RPM v.1 array harbours 14 viral and bacterial species. Arandom amplification protocol was used and in both studiesidentification not only at the species level but also at the strainlevel was obtained. This is of particular interest for surveillance ofepidemic outbreaks. The development of a second RPM chip (v.2) hasalready been initiated including 54 bacterial and viral species.However, the sensitivity and assay speed has to be improved to provide adiagnostic platform for pathogen detection.

The immediate precursor of a DNA array suitable in clinical diagnosticswas the reverse hybridisation line probe or blot. Line probe/blot assayshave been described for mutation detections and for genotyping and arealso commercially available (line probe assay (LiPA) from Innogenetics,Belgium). Generally, a generic amplification technology is used but upto now no studies have been published of line probe blots againstrespiratory viral pathogens. The microarray based assays allow highlycomplex analyses. However, they require specialized equipment andextensive post PCR handling.

(v) Beads or Microspheres Systems

In these products, detection is performed by a flow cytometer. In such asystem microspheres are internally dyed with two spectrally distinctfluorochromes. Using precise amounts of each of these fluorochromes, anarray is created consisting of 100 different microspheres sets withspecific spectral properties. Due to this different spectral property,microspheres can be combined, allowing up to 100 different targets to bemeasured simultaneously in a single reaction. For nucleic acid detectionusing microspheres, direct hybridisation of a labelled PCR amplifiedtarget DNA to microspheres bearing oligonucleotide capture probesproducts specific for each target sequence are used. Detection isperformed by two lasers, one to identify the distinct bead set and theother one to determine the specific target sequence.

Microsphere-based suspension array technologies, such as the Luminex®xMAP™ system, offer a new platform for high throughput multiplex nucleicacid testing. Compared to planar microarrays, they have the benefits offaster hybridisation kinetics and more flexibility in array preparation.Recently, a novel microsphere-based universal array platform, called theTag-It™ platform has been developed and used for detection anddifferentiation of 19 respiratory viruses. The Tag-It™ array platformfeatures universal, minimally cross-hybridizing tags for capturing thereaction products by hybridisation onto complementary anti-tag coupledmicrospheres. The respiratory viral panel on the Luminex platform wasdeveloped by TM Biosciences (Toronto, Canada) and validated onnasopharyngeal swabs and aspirates. An overall sensitivity of 96.1% wasobtained and data were confirmed by monoplex PCR and DFA. In addition 12double (out of 294 specimen samples) infections were detected. As withthe microarray based assays, these assays allow highly complex analysesbut require specialized equipment and extensive post PCR handling.

(vi) Mass Spectrometry Systems

These are assays wherein tags are released by UV irradiation andsubsequently analyzed by a mass spectrometer. Oligonucleotide primers,designed against conserved regions of the pathogen, are synthesized witha 5′ C6 spacer and aminohexyl modification and covalently conjugated bya photo-cleavable link to (Masscode) tags. A library of 64 differenttags has been established. Forward and reverse primers in individualprimer sets are labelled with distinct molecular tags. Amplification ofa particular pathogen target results in a dual signal in a massspectrometer that allows assessment of specificity.

Mass spectrometry is a homogeneous solution assay format that allows forsimultaneous detection of multiple nucleic acid sequences in a singlereaction thereby reducing time, labour and cost as compared tosingle-reaction-based detection platforms. A new class of molecularlabels, called cleavable mass spectrometry tags (CMSTs) has beendeveloped for simultaneous data acquisition. One application of CMSTtechnology is termed Masscode (Qiagen, Hilden, Germany) and is used fordifferential detections of respiratory pathogens. The general structureof CMSTs is highly modular and includes a photolabile linker, a massspectrometry sensitivity enhancer and a variable mass unit all connectedthrough a scaffold constructed around a central lysine residue. CMSTsare attached to the 5′-end of the oligonucleotide of the PCR primerthrough a photo-cleavable linker. The combination of the enhancer andthe variable mass unit specify the final mass of each individual CMST.Currently, a library of 64 distinct Masscode tags has been developed anda variety of mass spectrometry ionization methods can be applied. Agreat advantage of detection by mass spectrometry is the speed. Analysistakes only a few seconds.

Briese et al. (Emerg. Infect. Dis., 2005, 11; 310-313) developed adiagnostic assay comprising of 30 gene targets that represented 22respiratory pathogens. Nucleic acid from banked sputum, nasal swabs andpulmonary washes was tested and compared to virus isolation andconventional nested RT-PCR. Consistent results were obtained. Thedetection threshold was between 100-500 copies per sample. Massspectrometry is a very fast technique enabling highly complex analyses.However, this application requires specialized and expensive hardwarewhich, at the moment, is not common on a standard microbiologylaboratory.

The above described multiparameter approaches to identify anddifferentiate the causative agents of a RTI are a great step forward butstill have limitations. They take either too much time (>10 hours),require a lot of hands-on time, have a limited multi-parameter characterand/or need expensive equipment or large set-up costs to perform thetests.

SUMMARY OF THE INVENTION

Herein we describe a new multi-parameter approach to detect anddifferentiate various pathogens in a clinical sample.

The invention is in the technical field of detecting nucleic acidsequences in a sample, such as the detection of pathogenic organisms inclinical samples. More specifically, the invention relates to the fieldof detecting an infection caused by a pathogenic organism such as avirus or a bacterium in a clinical specimen by means of amplifying anddetecting specific nucleic acid sequences from said pathogenic organism.It provides a multiplex assay with the possibility to determine about 30different target nucleic acid sequences in a single one-tube assaycombined with real-time probe detection.

The invention relates to a method capable of simultaneously detecting aplurality of different target DNA templates in a sample, each DNAtemplate comprising a first target segment and a second target segment,the combination of both target segments being specific for a particulartarget DNA template, wherein the first and second target-specificsegments are essentially adjacent to one another and wherein the firsttarget segment is located 3′ from the second target segment, said methodcomprising the steps of:

-   -   a) an optional reverse transcription and/or pre-amplification        step,    -   b) bringing at least one DNA template into contact with a        plurality of different probe sets, each probe set being specific        for one target DNA template and allowing the at least one DNA        template to hybridise with a probe set specific for the at least        one DNA template,    -   c) forming a connected probe assembly comprising the specific        probe set,    -   d) amplifying the connected probe assembly to obtain at least        one amplicon,    -   e) detecting the presence of the at least one amplicon by        performing a real-time melting curve analysis,        wherein a donor or acceptor label is incorporated in the first        or second tag region, essentially adjacent to the detection        region and wherein step e) is performed by    -   providing a plurality of detection probes comprising    -   at least one fluorescent donor label or at least one acceptor        label complementary to the label incorporated in the first or        second tag region,    -   a nucleic acid region specifically hybridisable to said        detection sequence    -   allowing the at least one amplicon to hybridise with the        plurality of detection probes    -   monitoring hybridisation of the labelled detection probe at at        least one pre-selected temperature by measuring the fluorescence        of the acceptor label,    -   wherein said hybridisation of the labelled detection probe is        indicative for the presence of a target DNA template in the        sample.

Also, the invention relates to a kit for performing such a methodcomprising

-   -   a) A plurality of different probe sets    -   b) A source of a DNA ligase activity    -   c) A source of a DNA polymerase activity    -   d) At least one primer comprising at least one donor label    -   e) A plurality of detection probes comprising at least one        fluorescent label,    -   f) Instructions for performing the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic overview of the optional reverse transcriptionand pre-amplification step of the method according to the invention.

FIG. 2 shows a schematic overview of the probe sets used in the methodaccording to the invention and their hybridisation to the first andsecond target segments of the at least one DNA template.

FIG. 3 shows a schematic overview of a step wherein the probe sets areincorporated into a connected probe assembly. As a preferred example ofsuch a step it is shown how the hybridisation probes are connected bythe action of a ligase.

FIG. 4 provides a schematic overview of the formation of amplicons of aconnected probe assembly starting from 2 distinct DNA templates A and B.

FIG. 5 shows a schematic overview of real time PCR detection on aconnected probe assembly. Exemplified herein is the detection usinghybridisation probes.

FIG. 6 shows a schematic overview of a melting curve analysis employingtwo different probes X and Y with different melting temperatures. Thisresults in distinguishable signals in the melting curve analysis.

FIG. 7 shows a melt curve analysis of three channels of a clinicalsample containing human metapneumovirus. The human metapneumovirusdetection probe is labelled with Cy5 and has a theoretical meltingtemperature of 70° C.

FIG. 8 shows a melt curve analysis of two samples. Sample 1 containsMycoplasma pneumoniae and sample 2 contains respiratory syncytial virusA. Both samples are spiked with the internal amplification control.

FIG. 9 shows quantitative data of a sample containing Mycoplasmapneumoniae. Reaction 1 contains the undiluted sample, reaction 2contains the 10x diluted sample.

FIG. 10 shows an impression of a melt curve analysis of a samplecontaining a co-infection of influenza virus A virus and Chlamydiapneumoniae.

FIG. 11 shows a melt curve analysis of a OneTube reaction from a samplecontaining Mycoplasma pneumoniae.

FIG. 12 provides a schematic overview of the method according to thepresent invention, wherein the connected probe assembly is formed bysimultaneous hybridization of the first and second nucleic acid probeswith their corresponding target DNA template strands.

FIG. 13A shows an impression of a melt curve analysis of a samplecontaining a double infection of Influenza A and Rhino/Enterovirus. Theconnected probe assembly in this example is formed by simultaneoushybridization of the first and second nucleic acid probes with theircorresponding target DNA template strands.

FIG. 13B shows an impression of a melt curve analysis of a samplecontaining a double infection of Influenza A and Rhino/Enterovirus. Theconnected probe assembly in this example is formed by simultaneoushybridization of the first and second nucleic acid probes with theircorresponding target DNA template strands.

FIG. 13C shows an impression of a melt curve analysis of a samplecontaining a double infection of Influenza A and Rhino/Enterovirus. Theconnected probe assembly in this example is formed by simultaneoushybridization of the first and second nucleic acid probes with theircorresponding target DNA template strands.

FIG. 14 shows an impression of a melt curve analysis of a samplecontaining a co-infection of Adenovirus and Influenza A. The connectedprobe assembly in this example is formed by simultaneous hybridizationof the first and second nucleic acid probes with their correspondingtarget DNA template strands.

FIG. 15A shows an impression of a melt curve analysis of a reaction of asample negative for pathogens; the negative result is validated by thepresence of the Internal Control, and a different Amplification Controlto distinguish this reaction from that of FIG. 15B.

FIG. 15B shows an impression of a melt curve analysis of a reaction of asample negative for pathogens; the negative result is validated by thepresence of the Internal Control, and a different Amplification Controlto distinguish this reaction from that of FIG. 15A.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to a method capable ofsimultaneously detecting a plurality of different target DNA templatesin a sample, each DNA template comprising a first target segment and asecond target segment, the combination of both target segments beingspecific for a particular target DNA template, wherein the first andsecond target-specific segments are essentially adjacent to one anotherand wherein the first target segment is located 3′ from the secondtarget segment, said method comprising the steps of:

-   -   a) an optional reverse transcription and/or pre-amplification        step,    -   b) bringing at least one DNA template into contact with a        plurality of different probe sets, each probe set being specific        for one target DNA template and allowing the at least one DNA        template to hybridise with a probe set specific for the at least        one DNA template,    -   c) forming a connected probe assembly comprising the specific        probe set,    -   d) amplifying the connected probe assembly to obtain at least        one amplicon,    -   e) detecting the presence of the at least one amplicon by        performing a real-time melting curve analysis

Such a method has been disclosed in EP1130113A1. Therein the detectionof an amplicon obtained by multiplex ligation dependent amplification(MLDA) is described by performing a real time melting curve analysis. Inthat kind of analysis, a number of different stuffer fragments areprovided that result in a different melting behaviour of the ampliconsthemselves. Such amplicons may then for instance differ in length, whichmakes them amenable for detection using simple gel-electrophoresis.Alternatively, amplicons may also be detected using the 5′ nucleaseactivity of some polymerases (Taqman®). Other real time detectionmethods are disclosed that do not rely on the destruction ofoligonucleotides but instead rely on the use of molecular beacons. Suchdetection requires a probe containing a fluorophor and a quencher. Athird alternative disclosed in EP 1130113 is the use of detection probesconsisting of two entities, each being complementary to sequencespresent on the amplicon each containing a fluorescent moiety whereinfluorescent resonance energy transfer occurs upon binding of both probeentities to the amplicon.

It has now been found that another way of detecting the ampliconprovides a more reliable and robust assay that allows for the truemultiplexing of up to 30 different target sequences even in a one-tubeor two-tube system. This is also referred herein as a closed system asopposed to the above prior art which is inherently an open system. Themethod according to the invention provides much more freedom to engineerprobe assemblies and therefore results in a more reliable assay capableof distinguishing better between a large number of different amplicons.

The invention relates to a method capable of simultaneously detecting aplurality of different target DNA templates in a sample, each DNAtemplate comprising a first target segment and a second target segment,the combination of both target segments being specific for a particulartarget DNA template, wherein the first and second target-specificsegments are essentially adjacent to one another and wherein the firsttarget segment is located 3′ from the second target segment, said methodcomprising the steps of:

-   -   a) an optional reverse transcription and/or pre-amplification        step,    -   b) bringing at least one DNA template into contact with a        plurality of different probe sets, each probe set being specific        for one target DNA template and allowing the at least one DNA        template to hybridise with a probe set specific for the at least        one DNA template,    -   c) forming a connected probe assembly comprising the specific        probe set,    -   d) amplifying the connected probe assembly to obtain at least        one amplicon,    -   e) detecting the presence of the at least one amplicon by        performing a real-time melting curve analysis,        wherein a donor or acceptor label is incorporated in the first        or second tag region, essentially adjacent to the detection        region and wherein step e) is performed by    -   providing a plurality of detection probes comprising    -   at least one fluorescent donor label or at least one acceptor        label complementary to the label incorporated in the first or        second tag region,    -   a nucleic acid region specifically hybridisable to said        detection sequence    -   allowing the at least one amplicon to hybridise with the        plurality of detection probes    -   monitoring hybridisation of the labelled detection probe at at        least one pre-selected temperature by measuring the fluorescence        of the acceptor label,        wherein said hybridisation of the labelled detection probe is        indicative for the presence of a target DNA template in the        sample.

The method according to the invention is capable of simultaneouslydetecting a plurality of different target DNA templates in a sample.This means that the method has the potential of detecting more than onetarget DNA template in a sample at the same time. If the sample containsonly one target DNA template, then the method of course detects that onetemplate, the probes specific for other templates are then not used.

In many clinical samples, sufficient copies of DNA templates areavailable to perform the method according to the invention without theoptional pre-amplification step.

In some clinical samples, however, insufficient copies of a DNA templatemay be available. In such case, an optional pre-amplification step hasto be performed. Also, when the template is an RNA template, this has tobe converted into a DNA template by a reverse transcription step. Boththese steps are known in the art and the skilled person will be aware ofways to perform them. A schematic overview of this step is provided inFIG. 1. Additional guidance may be found in Sambrook et al., 2000.Molecular Cloning: A Laboratory Manual (Third Edition) Cold SpringHarbor Laboratory Press.

The method according to the invention is then performed by bringing atleast one DNA template into contact with a plurality of different probesets (FIG. 2). This plurality of probe sets is a predetermined set ofprobes that are specific for a particular set of DNA templates. Anadvantageous choice of probe sets may be based upon the diversity ofagents that are often found together in a clinical disease. For example,for the screening and typing of respiratory tract infections it may beadvantageous to combine probe sets specific for influenza virus A and B,parainfluenza virus 1, 2, 3 and 4, respiratory syncytial virus A and B,rhinovirus, coronavirus 229E, OC43 and NL63, human metapneumovirus,adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionellapneumophila and Bordetella pertussis. Also, the method may beadvantageously employed in the detection of infections, like infectionsin blood. For example, for the screening and typing of blood associatedviruses it may be advantageous to combine probe sets for thesimultaneous detection of human immunodeficiency virus, hepatitis Bvirus and hepatitis C virus.

If one or more of the DNA templates for which the probe sets aredesigned is present in the sample, then the probe set specific for thetarget DNA template will specifically hybridise with the first andsecond target segments of the DNA template (FIG. 2).

The skilled person will be aware of the constraints that apply whenselecting a suitable region on the DNA template that could serve asfirst and second target segments. Most importantly, this should be aconserved region so that the natural variability of the template doesnot cause false-negative results. Additional guidance in the choice ofthe target regions is to be found in Schouten et al., WO 01/61033,Schouten et al., Nucl. Acids Res. 2002, vol 30 No 12; e57 and Sambrooket al., 2000. Molecular Cloning: A Laboratory Manual (Third Edition)Cold Spring Harbor Laboratory Press.

The specific probe set is then allowed to form a connected probeassembly (FIG. 3, 12). This may be accomplished by a ligase chainreaction which results in multiple copies of the connected probeassembly or by a single ligase step (as depicted in FIG. 3) followed byamplification of the connected probe assembly (FIG. 4). Alternatively,the connected probe assembly may be formed by simultaneous hybridizationof the first and second nucleic acid probes with their correspondingtarget DNA template strands (FIG. 12). Herein, the first nucleic acidprobe of each probe set is allowed to hybridize to a first target DNAtemplate strand, and the second nucleic acid probe of each probe set isallowed to hybridize to its opposite target DNA template strand. Aftersubsequent hybridization of the amplification primers, to the tagregions of said probes, the connected probe assembly is then amplified.

When performing a single ligase step, it may be advantageous to choose atemperature that is not too low, i.e. between 50 and 65° C. T4 ligaseperforms at temperatures between 42 and 47° C. and is therefore lesssuitable when a high specificity of the assay is required. It is alsoadvantageous to use the smallest possible volume. Temperature-stabileand temperature-labile ligases are equally suitable. Additional guidancein the choice of suitable ligase enzymes and conditions for its use isto be found in Schouten et al., WO 01/61033, Schouten et al., Nucl.Acids Res. 2002, vol 30 No 12; e57 and Sambrook et al., 2000. MolecularCloning: A Laboratory Manual (Third Edition) Cold Spring HarborLaboratory Press.

The detection of the amplified connected probe assembly isadvantageously performed in real time (FIG. 5), preferably in a closedsystem, for instance using fluorescence resonance energy transfer (FRET)probes in a real-time PCR apparatus. To this end, a melting curveanalysis may be performed (FIG. 6). In that case, fluorescence ismonitored with increasing temperature, a decrease in fluorescence isobtained when probes melt off (For a survey see Mackay et al 2002,Nucleic Acids Res. 30; 1292-1305).

The method according to the invention has a superior sensitivity, evenwhen compared to MLPA. Standard MLPA requires approximately 6000 singlecopy targets to obtain reproducible results. Samples originating frompatients suffering from viral and bacterial infections contain onaverage much less copies.

In more detail, the invention relates to a method as described abovewherein step b) is performed by bringing at least one DNA template intocontact with a plurality of different probe sets, each probe set beingspecific for one target DNA template and allowing the at least one DNAtemplate to hybridise with a probe set specific for the at least one DNAtemplate and each probe set comprising:

-   -   a first nucleic acid probe having    -   a first target region hybridisable to the first target segment    -   a first tag region, 5′ from the first target region comprising a        first tag sequence    -   a second nucleic acid probe having    -   a second target region hybridisable to the second target segment    -   a second tag region, 3′ from the second target region comprising        a second tag sequence    -   wherein at least one of the first and second nucleic acid probes        contains a detection sequence located 5′ from the second tag        sequence or located 3′ from the first tag sequence.

The invention also relates to a method as described above, wherein stepc) is performed by allowing the first and second nucleic acid probes tocovalently connect to one another if hybridised to said target DNAtemplate, thereby forming at least one connected probe assembly flankedby the first and second tag regions.

The invention also relates to a method as described above, wherein stepc) is performed by allowing the first and second nucleic acid probes tohybridize to their corresponding target DNA template, hence forming atleast one connected probe assembly comprising the target DNA templateand the first and second nucleic acid probes, flanked by theircorresponding first and second tag regions. Herein, the first nucleicacid probe of each probe set is allowed to hybridize to a first targetDNA template strand, and the second nucleic acid probe of each probe setis allowed to hybridize to its opposite target DNA template strand.

The invention also relates to a method as described above, wherein stepd) is performed by:

-   -   allowing the at least one connected probe assembly to contact        with a nucleic acid primer pair comprising primer 1 and primer        2, wherein    -   primer 1 comprises a first nucleic acid sequence hybridisable to        the complement of the first tag sequence and    -   primer 2 comprises a second nucleic acid sequence hybridisable        to the second tag sequence    -   amplification of said at least one connected probe assembly in        order to obtain at least one amplicon comprising    -   the first tag region or at least part thereof    -   the first target region    -   the second target region    -   the detection region    -   the second tag region or at least part thereof        or the complements thereof.

The invention also relates to a method as described above, wherein stepe) is performed by:

-   -   detecting the presence of said at least one amplicon by    -   providing a plurality of detection probes comprising    -   at least one fluorescent label    -   a nucleic acid region specifically hybridisable to said        detection sequence    -   allowing the at least one amplicon to hybridise with the        plurality of detection probes    -   monitoring hybridisation of the detection probe at at least one        pre-selected temperature by measuring the fluorescence of the        label,        wherein said hybridisation of the detection probe is indicative        for the presence of a target DNA template in the sample.

The phrase “simultaneously detecting” as used herein indicates that aplurality, i.e. more than 1, different targets may be detected in oneand the same analysis. For that purpose, the method according to theinvention provides a different pair of target regions for each nucleicacid template to be amplified. This presupposes that at least part ofthe nucleic acid sequence of the target DNA template, such as a DNA orRNA virus or the genome of a bacterium, is known. From that knownnucleic acid sequence, the skilled person may choose suitable first andsecond target segments for hybridisation of specific probes. The firstand second target segments are preferably long enough to allowhybridisation and annealing of the probes at elevated temperatures,typically about 20 to 40 nucleic acids. The skilled person will takecare that the first and second target segments are sufficientlydifferent from each other to allow specific hybridisation with the probeset. For that same reason, the skilled person will choose sufficientlydifferent first and second target segments from the various nucleic acidtemplates that are to be detected. Means and methods for doing that arereadily available in the art and known to the skilled person. Additionalguidance in the choice of the target sequences is to be found inSchouten et al., WO 01/61033, Schouten et al., Nucl. Acids Res. 2002,vol 30 No 12; e57 and Sambrook et al., 2000. Molecular Cloning: ALaboratory Manual (Third Edition) Cold Spring Harbor Laboratory Press.

The first and second target segments are chosen essentially adjacent.That means that the first and second nucleic acid probes that hybridiseto these regions are positioned such that they may easily couplecovalently when a suitable ligase is present. To allow connection ofessentially adjacent probes through ligation, one possibility is togenerate probes that leave no gap upon hybridisation. However, it isalso possible to provide at least one additional single stranded nucleicacid complementary to at least one interadjacent part of said targetnucleic acid, whereby hybridisation of said additional nucleic acid tosaid interadjacent part allows the connecting of two adjacent probes. Inthis embodiment of the invention a gap upon hybridisation of the probesto the target nucleic acid is filled through the hybridisation of saidadditional single stranded nucleic acid. Upon connecting andamplification the resulting amplicon will comprise the sequence of saidadditional single stranded nucleic acid. One may choose to have saidinteradjacent part to be relatively small thus creating an increaseddifference in the hybridisation efficiency between said oneinteradjacent part of said target nucleic acid and a nucleic acid thatcomprises homology with said one interadjacent part of said targetnucleic acid, but comprises a sequence which diverges from in one ormore nucleotides. In another embodiment of the invention a gap betweenprobes on said target nucleic acid is filled through extending a 3′ endof a hybridised probe or an additional nucleic acid filling part of aninteradjacent part, prior to said connecting.

As used herein, the term “complementary” in the context of nucleic acidhybridisation or “complementary nucleic acid” indicates a nucleic acidcapable of hybridising to another nucleic acid under normalhybridisation conditions. It may comprise mismatches at a small minorityof the sites.

The term “complementary” in the context of a fluorescent label refers toeither a donor or acceptor label. A donor label is complementary to anacceptor label and vice versa.

As used herein, “oligonucleotide” indicates any short segment of nucleicacid having a length between 10 up to at least 800 nucleotides.Oligonucleotides can be generated in any matter, including chemicalsynthesis, restriction endonuclease digestion of plasmids or phage DNA,DNA replication, reverse transcription, or a combination thereof. One ormore of the nucleotides can be modified e.g. by addition of a methylgroup, a biotin or digoxigenin moiety, a fluorescent tag or by usingradioactive nucleotides.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid sequence synthesis when placed under conditions in whichsynthesis of a primer extension product which is complementary to anucleic acid strand is induced, i.e. in the presence of differentnucleotide triphosphates and a polymerase in an appropriate buffer(“buffer” includes pH, ionic strength, cofactors etc.) and at a suitabletemperature. One or more of the nucleotides of the primer can bemodified for instance by addition of a methyl group, a biotin ordigoxigenin moiety, a fluorescent tag or by using radioactivenucleotides.

A primer sequence need not reflect the exact sequence of the template.For example, a non-complementary nucleotide fragment may be attached tothe 5′ end of the primer, with the remainder of the primer sequencebeing substantially complementary to the strand.

The amplicon obtained in step d) above advantageously comprises thefirst and second tag regions. Alternatively, the amplicon may alsocontain only part of the first and second tag regions, depending on thelength of the primer pair used. The minimum length of said part of thefirst and second tag regions as contained in the amplicon corresponds tothe lengths of the primers used.

As used herein, the term “target sequence” refers to a specific nucleicacid sequence to be detected and/or quantified in the sample to beanalysed.

As used herein, the term “hot-start” refers to methods used to preventpolymerase activity in amplification reactions until a certaintemperature is reached.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein the term “PCR” refers to the polymerase chain reaction(Mulis et al U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). The PCRamplification process results in the exponential increase of discreteDNA fragments whose length is defined by the 5′ ends of theoligonucleotide primers.

As used herein, the terms “hybridisation” and “annealing” are used inreference to the pairing of complementary nucleic acids.

Conventional techniques of molecular biology and recombinant DNAtechniques, which are in the skill of the art, are explained fully inthe literature. See, for instance, Sambrook, Fritsch and Maniatis,Molecular Cloning; A Laboratory Manual, Second Edition (1989) and aseries, Methods in Enzymology (Academic Press, Inc.) and Sambrook etal., 2000. Molecular Cloning: A Laboratory Manual (Third Edition) ColdSpring Harbor Laboratory Press.

The description of the method according to the invention should not beinterpreted so narrowly as that it could not detect other nucleic acidtemplates, such as RNA templates. A skilled person would understand thedisclosure of the present invention as to include the amplification ofRNA sequences such as RNA templates, for instance by introducing apre-amplification step in order to generate samples containing DNAtemplates corresponding to RNA sequences in a sample such as a clinicalsample. The thus obtained DNA samples could then be used in a method asdescribed above.

Therefore, a method according to the invention as disclosed hereinallows for the detection of a plurality of different DNA templates thatmay be obtained by amplifying a plurality of different nucleic acidssuch as DNA or RNA in a sample, such as for instance a clinical sample.

Such a method may be particularly advantageous when the plurality ofdifferent nucleic acids is derived from micro-organisms, such asbacteria, viruses, algae, parasites, yeasts and fungi. If suchmicro-organisms are pathogenic, a quick and reliable method that candetect a variety of different nucleic acid templates may be particularlyadvantageous.

In many circumstances, a sample may contain substances that interferewith a subsequent amplification and/or detection step. In a methodaccording to the invention, such interference may be avoided byextracting the plurality of DNA templates from a sample beforehybridisation. Hence, the invention relates to a method as describedabove, wherein the plurality of DNA templates is extracted from thesample before allowing the DNA templates to hybridise with a pluralityof different probes.

In a method according to the invention, tag sequences contained in thefirst and second nucleic acid probes are used to amplify the first andsecond target-specific sequences. It may be advantageous when these tagsequences are chosen in such a way that they do not hybridise with anyof the target DNA templates that are to be detected. It should be notedthat such is not mandatory and that the method may as well be performedwithout that modification. However sensitivity and specificity of themethod are improved in a method as described above wherein at least oneof the first and second tag sequences have a nucleic acid sequencechosen in such a way that the first and second tag sequences do nothybridise to the plurality of different target sequences. Typically, asuitable tag sequence has a G/C content of about 50, a length of betweenapproximately 20 to 30 nucleotides and a Tm of 60 to 80° C.

In order to improve specificity and sensitivity of the method, it mayalso be particularly advantageous that not only the first and/or secondtag sequence does not hybridise with any of the target DNA templatesthat are to be detected, but that the entire tag region does nothybridise. Hence, a method according to the invention relates to amethod as described above wherein at least one of the first and secondtag regions have a nucleic acid sequence chosen in such a way that thefirst and second tag regions do not hybridise to the plurality ofdifferent target sequences.

The tag sequences in this embodiment of the method according to theinvention serve the purpose of amplifying the connected probeassemblies. The tag sequences of the plurality of different probe setsmay all be different; however, for reasons of convenience, the tagsequences may advantageously be universal, so that each differentconnected probe assembly may be amplified with one and the same nucleicacid primer pair. Hence, in one embodiment, the invention relates to amethod as described above wherein at least one of the first and secondtag sequences is a universal sequence. For the avoidance of doubt, bythe above description it is meant that all first tag sequences areidentical and that all second tag sequences are identical, notnecessarily that the first and second sequences are identical, althoughthat may also be the case without affecting the usefulness of themethod.

When both oligonucleotides to be ligated are hybridised to the targetnucleic acid, a covalent phosphate link between the two fragments may beformed enzymatically by a ligase. Although other methods of covalentlycoupling two nucleic acids are available (such as the ligase chainreaction) a method according to the invention is most advantageouslyperformed when the first and second probes are attached to one anotherby a ligase. In a particular embodiment, the invention therefore relatesto a method as described above, wherein the first and second nucleicacid probes are covalently connected to each other in order to form saidat least one connected probe assembly by an enzyme having ligaseactivity.

In a particular embodiment, the invention also relates to a method asdescribed above, wherein the first and second nucleic acid probes arehybridized to their corresponding DNA target template to form said atleast one connected probe assembly. Herein, the first nucleic acid probeof each probe set is allowed to hybridize to a first target DNA templatestrand, and the second nucleic acid probe of each probe set is allowedto hybridize to its opposite target DNA template strand.

In one embodiment, probes may be used that hybridise to the templatespatially close to each other but not adjacent enough to allow immediateligation of the probes. In that case, the probe with the target specificsequence at its 3′ end can be elongated by a polymerase in the presenceof a suitable buffer and the four dNTP's in order to make ligation ofthe two probes possible. As an alternative the gap between the probescan be filled by complementary oligonucleotides that can be ligated tothe probes.

DNA ligases are enzymes capable of forming a covalent phosphate linkbetween two oligonucleotides bound at adjacent sites on a complementarystrand. These enzymes use NAD or ATP as a cofactor to seal nicks indouble stranded DNA. Alternatively chemical autoligation of modifiedDNA-ends can be used to ligate two oligonucleotides bound at adjacentsites on a complementary strand (Xu, Y. & Kool, E. T. (1999), NucleicAcid Res. 27, 875-881).

It can also be envisaged that a method according to the invention may beperformed by RNA or DNA primers. DNA primers are more often used forreasons of convenience and because they are more stable than RNAprimers. Therefore the invention relates to a method as described abovewherein at least one of the nucleic acid primers 1 and primers 2 is aDNA primer.

The detection sequence may be located anywhere between the two tagsequences; however, if the detection sequence is designed to be locatedessentially adjacent to the first or second tag sequence, it has theleast chance of interfering with the hybridisation and/or ligationreactions. This positioning of the detection sequence has the additionaladvantage that the detection sequence may be used in a detectionreaction involving FRET probes. Hence, a method according to theinvention may be characterised as a method as described above whereinthe detection sequence is essentially adjacent to the first or secondtag sequence. In this respect, the term “essentially adjacent” is meantto indicate that energy transfer may occur when an end-labelled probethat hybridises to the detection sequence is capable of energy transferwith a label at the end of the tag sequence. It is even more preferredthat the detection sequence is immediately adjacent to the second tagsequence. In the latter case the energy transfer from an internallylabelled probe assembly to the fluorescently labelled detection probe ismost efficient. In general, energy transfer is optimal when the distancebetween the labels is less than 5 to 10 nucleotides, preferably lessthan 5 such as 4, 3, 2, 1 or 0.

The detection sequence may be a random sequence chosen in such a waythat it does not interfere with any of the other reagents used in themethod, except for the detection probe. Advantageously, the detectionsequence is chosen in such a way that the Tm of the detection probe isbetween 50 and 75° C.

Such a detection method requires the use of internally labelledamplified regions or amplicons. Such amplicons may be obtained byproviding labelled primers in a method according to the invention. Theseprimers may then be labelled with a donor or acceptor fluorescent label;conversely the fluorescently labelled detection probe should then ofcourse contain a complementary donor or acceptor fluorescent label.Hence, the invention also relates to a method as described above whereinat least one of primers 1 or primers 2 comprises at least one internaldonor or acceptor fluorescent label at or near its 3′ end therebyproviding at least one internally labelled amplicon upon amplificationof said at least one connected probe assembly. It is most preferred tohave the label situated at the 3′ end of the primer since it will thenbe in the closest contact with the complementary label of the detectionprobe. Examples are provided of a method wherein primer 2 comprises theinternal label at its 3′ end. Also, a method is exemplified wherein saidat least one internally labelled amplicon is detected by said pluralityof detection probes provided with at least one complementary donor oracceptor fluorescent label at or near its 3′ end.

In order to detect the adjacent fluorescent molecules, the donorfluorescent label may be excited and the fluorescence of the acceptormay be measured. The non-adjacent donor and acceptor labels that arestill in the reaction vessel do not contribute to the signal because thedistance between the donor and the acceptor is too large. In that way,only those DNA templates are detected that allowed the formation of aninternally labelled probe assembly (FIG. 5). The method according to theinvention thus allows simultaneous real-time detection of a plurality ofdifferent DNA templates. A method according to the invention may thus becharacterised as a method as described above, wherein the detection ofat least one internally labelled amplicon comprises the step of excitingthe donor fluorescent label and measuring the fluorescence of theacceptor fluorescent label.

In a particular embodiment, a method according to the invention alsoprovides the opportunity to distinguish a plurality of connected probeassemblies by choosing detection probes in such a way that they can bedistinguished by their difference in melting temperature when hybridisedto the detection sequence. Such may be accomplished by designingdetection probes with a different length or nucleotide composition.Hence, the invention relates to a method as described above, whereinsaid plurality of detection probes is chosen in such a way that theindividual detection probes can be distinguished from each other bytheir difference in melting temperature when hybridised to the detectionsequence.

This embodiment multiples the number of target DNA templates that may bedetected. In a real-time assay; melting curves of probes that are only afew degrees Celsius apart may be easily distinguished. Herein weexemplify the use of probes with melting temperatures that are 3 to 5degrees Celsius apart. Hence, in an advantageous embodiment, theinvention relates to a method as described above, wherein the at leastone pre-selected temperature consists of a temperature range of at least3 degrees Celsius and wherein a melting curve analysis is performed todetect a decrease in fluorescence when a detection probe de-hybridises.

In order to further increase the number of target DNA templates that maybe detected, different fluorescent labels may be employed. Hence, theinvention relates to a method as described above, wherein differentfluorescent labels are used to increase the number of different targetDNA templates to be detected.

To monitor the efficiency of nucleic acid extraction from the primarysample, and to monitor the efficiency of a possible pre-amplificationreaction, an internal control (IC) may be added to the primary sampleprior to extraction. The IC should contain a nucleic acid of a knownsequence that is not present in any of the pathogens to be detected, norin any of the other sequences possibly present in the primary sample,The IC sequence is detected in the same way as the pathogens possiblypresent in the sample. If the sample is negative for the pathogenspossibly detected in the assay, the IC should still be positive toensure (i) adequacy of the nucleic acid extraction from the sample, and(ii) proper progress of the reaction when performed. Hence, negativeresults are validated. The amount of IC added to the sample is adjustedin such a way that false negative results due to out-competition of alow amount of nucleic acid from a pathogen by the IC are ruled out.Consequently, if a pathogen is present in the sample, out-competition ofIC may occur and is allowed, and the reaction is considered valid when apathogen is detected but the IC is not.

If the number of target sequences to be detected exceeds themultiplexing capacity that can be obtained by the maximum number ofdifferent melting temperatures and/or the maximum number of fluorescentlabels available, the number of reactions performed with a particularsample may be doubled, tripled, quadrupled etc. etc., which results in adouble, tripled, quadrupled etc multiplexing capacity. The number ofmultiplex reactions may follow a pre-amplification step that containedpre-amplification primers for all pathogens detected in all reactions.Alternatively, if the desired multiplexing exceeds the multiplexingcapacity of the pre-amplification step, this pre-amplification step toomay be split up into more than one separate reaction, each containingone or more pathogen-specific primer pairs.

To simplify both the manufacturing and the interpretation of the meltingpeaks, the fluorescent labels and melting temperatures of the detectionprobes may be identical in each of the final reactions, while a givendetection sequence may be linked to different target-specific sequencesin the different reactions. Also, the primers 1 and 2 that directamplification of said primary reaction product may be identical betweenthe different reactions. To prevent the user from mixing up thedifferent reactions which would result in misdiagnosis, each reactionmay contain a synthetic nucleic acid (from here on referred to asamplification control (AC)) containing the first and second tag region,and a detection region that differs between each reaction, resulting indifferent melting temperatures for these amplification controls by meansof which the different reactions can be distinguished. In addition, theAC(s) may contain part random or non-pathogenic sequence when necessary.

When multiple final reactions are performed following a singlepre-amplification step, a pathogen is present in a sample, and completeout-competition of the IC takes place during the pre-amplification stepas described above, the IC signal will be absent from each finalreaction. Hence, a positive signal will only be observed in theparticular reaction mix that contains the inner primers specific for thepathogen present, and other reaction mixes may show no signal at all.From the absence of signals from the other reaction mixes,out-competition of the IC and improper reaction progress due to othercauses cannot be distinguished. However, the AC signal present in thereaction mixes ensures proper progress of this reaction. The ACs areonly present during the reaction and not during the pre-amplificationstep; therefore they are not sensitive to possible out-competitionduring the pre-amplification step.

In a certain embodiment, a software tool may be provided with the kit toallow unequivocal and user-independent interpretation of melting peaks,and automated assignment of the pathogen associated with the detectedmelting peaks. When using a software tool in the case of abovementionedmultiple reaction mixes, the software tool should be able to detectwhich reaction mix is contained in a particular sample position. This isenabled by abovementioned ACs that show different melting peaks in eachreaction.

In order to avoid contamination, it may be advantageous to limit thenumber of times that a reaction vessel has to be opened before the finaldetection takes place. Due to the specific number and sequence of stepsas well as the reaction conditions employed in the present invention, wewere able to limit the number of consecutive steps to at most two (thepre-amplification step and the actual reaction). In a preferredembodiment, the method according to the invention may even be performedin a single, closed reaction vessel, thus eliminating any risk ofcontamination. Hence, the invention relates to a method as describedabove wherein the hybridisation and ligation steps are performed in onereaction vessel. Alternatively, the invention relates to a method asdescribed above wherein the pre-amplification and actual reaction areperformed in one reaction vessel.

The present inventors have discovered that the first hybridisation stepof the present method may be performed under low salt concentrations andin the presence of Mg ions, preferably MgCl₂, thereby allowing theligation reaction to be performed in the same reaction vessel as thehybridisation reaction. More in particular, the invention relates to amethod as described above wherein the hybridisation step is performed inthe presence of Mg ions and in the presence of less than 200 mM KCl.

Also, the amplification and ligation step may be performed in onereaction vessel.

If conditions are even further optimised, the entire reaction may beperformed even in one reaction vessel. For such a one tube reaction, thefollowing conditions are advantageous. For the amplification step,preferably a hot-start enzyme is used. This has the advantage ofproviding a longer activation period so that the amplification processallows sufficient time for the hybridisation and ligation steps withoutinterference with the amplification step. This further adds to thereliability of the method and allows for an even increased sensitivity.Hence, the invention relates to a method as described above wherein ahot-start enzyme is used for the amplification step.

A one-tube or one-step reaction is also often referred to as a closedsystem. This term is used to indicate that a system produces ampliconswithout the need to reopen the vessel for the addition of reagents, itmay also indicate that amplicons are detected in the same vessel withoutthe need to re-open the vessel after the amplification step. Preferablyit refers to a system that produces and detects amplicons without theneed to open the vessel only once after the addition of the startingreagents. In the context of the present invention, a one-step reactionrefers to a reaction wherein at least steps b, c, d and e are performedin one reaction vessel without the need to open the vessel in betweenthese steps, e.g. for the addition or removal of reaction compounds.

A two-tube or two-step reaction may be considered as a semi-closedsystem. This term indicates that the reaction as described above may beperformed in a single reaction vessel wherein the reaction vessel isopened only once in between steps b to e. This is preferably done inbetween steps b and c.

Mg concentration for the one step assay are preferably chosen between0.5 and 5 mM and NaCl concentrations are optimal between 0 and 250 mM,preferably between 25 and 150 mM. It is also advantageous when thevolume is kept as small as possible, such as between 5 and 25microliter.

Hence, the invention relates to a method as described above, wherein thehybridisation, ligation and amplification steps are performed in onereaction vessel.

It even proved possible to perform the entire method in a singlereaction vessel, i.e. the hybridisation, ligation, amplification anddetection step could be combined without the need of opening thereaction vessel even only once. Hence, the invention relates to a methodas described above, wherein the detection step is performed in the samevessel as the amplification step.

A method according to the invention may be advantageously employed inany application in which pathogens e.g. viruses, bacteria, fungi,yeasts, algae, protozoa and multicellular parasites, and normal andmutated host sequences and any combination of these, have to be detectedand/or identified. It may also be employed in any application in whichpathogens e.g. viruses, bacteria, fungi, parasites have to be typed. Itmay also be employed in any application in which pathogens e.g. viruses,bacteria, fungi, parasites, or hosts have to be screened for specificgenetic properties.

Specific applications may be found in the screening and typing ofPlasmodium species in blood of Malaria patients, the detection andidentification of Anopheles species and the determination of their hostpreference and screening for sporozoites of Plasmodium species, thescreening and typing of tick bites associated species like Ehrlichia andAnaplasma species, the screening and typing of pathogens associated withfetal abnormalities such as cytomegalovirus (CMV) and other humanherpesviruses (HHVs), Parvovirus B19, Toxoplasma gondii, Treponemapallidum and rubella virus in pregnant women, the screening and typingof Clostridium difficile, the screening and typing ofmeningitis-associated pathogens like HHV1 through 8, enterovirus,parecho virus Listeria monocytogenes, Staphylococcus species,Haemophilus influenzae, Streptococcus pneumoniae, Streptococcusagalactiae, Neisseria meningitidis, Borrelia burgdorferi sensu lato,Klebsiella pneumonia K1, Cryptococcus neoformans, Cryptococcus gattiiand Mycobacterium tuberculosis in cerebrospinal fluid, the screening andtyping of virus infections like human herpesviruses (HHVs) and EpsteinBarr virus (EBV) in cord blood, the screening and typing of humanenteric viruses in groundwater, the screening and typing of blood-borneviruses like human immunodeficiency virus, hepatitis B virus andhepatitis C virus, the screening and (sub)typing of influenza viruses,the screening for resistance associated polymorphisms in humanimmunodeficiency viruses and hepatitis viruses, the screening and typingof mycobacterium species and identification of the Mycobacteriumtuberculosis complex members, the screening and typing of humanpapillomaviruses, the screening and typing of viruses like rice tungrobaccilliform virus, rice tungro spherical virus on rice plants affectedby rice tungro disease, the screening and typing of Bordetella pertussisstrains and screening for specific genetic properties, the screening andtyping of viruses, bacteria and parasites like human papillomavirus,Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium,Trichomonas vaginalis, Treponema pallidum, commonly associated withsexual transmitted diseases, the screening and typing of Dermatophytespecies commonly associated with dermatophytosis, the screening andtyping of methicillin resistant Staphylococcus aureus, the screening andtyping of pathogens like Salmonella spp., Campylobacter spp., Norovirus,commonly associated with foodborne infections and toxications, thescreening and typing of pathogens commonly associated with respiratoryinfections in cattle and pigs, the screening and typing of viruses likenoroviruses, rotaviruses, astroviruses, hepatitis A viruses andenteroviruses in oyster samples, the screening and typing of bacterialike mutant streptococci and lactobacilli, commonly associated withdental caries, the screening and typing of tropical diseases (e.g.African trypanosomiasis, dengue fever, leishmaniasis, schistosomiasis,Chagas' disease, leprosy, lymphatic filariasis, cholera, yellow feveretc.), the screening and typing of zoonoses (e.g. salmonellosis andcampylobacteriosis, brucellosis, Rabies, leptospirosis, shigellosis,echinococcosis, toxoplasmosis etc.), the screening and typing ofpathogens commonly associated with gastroenteritis (e.g. rotaviruses,noroviruses, adenoviruses, sapoviruses, astroviruses etc.), and thescreening and typing of multidrug resistant bacterial strains.

Diagnostic methods for the detection of DNA templates derived frompathogens are often provided in kits. Hence the invention also relatesto a kit for performing a method as described above comprising

-   -   a) A plurality of different probe sets    -   b) A source of a DNA ligase activity    -   c) A source of a DNA polymerase activity    -   d) At least one primer comprising at least one donor label    -   e) A plurality of detection probes comprising at least one        fluorescent label,    -   f) Instructions for performing the method.

Depending on the particular embodiment chosen, the kit may compriseadditional probes, primers, labelled and unlabelled, as the particularembodiment requires. A particular advantageous kit contains detectionprobes with different melting temperatures, preferably these detectionprobes have melting temperatures that differ at least 3 degrees Celsiusfrom each other.

EXAMPLES

The following examples are provided to aid the understanding of thepresent invention without the intent to limit the invention. It is nowwithin the reach of the skilled artisan to make modifications in theprocedures set forth without departing from the spirit of the invention.

In the following examples, a method capable of simultaneously detectinga plurality of different targets in a clinical sample is described.These specific examples, when at least partially combined, form an assayaccording to the invention that detects a plurality of differentpathogens or disease agents in a clinical sample, in this case anasopharyngeal lavage. The method is capable of simultaneously detectingat least two disease agents selected from the group consisting ofinfluenza A and B, respiratory syncytial virus A and B, humanmetapneumovirus, Chlamydia pneumoniae, Mycoplasma pneumoniae andLegionella pneumophila.

Examples 1-9 represent the experimental set-up and results for a methodaccording to a first embodiment of the present invention, wherein theconnected probe assembly is formed by ligation of the first and secondnucleic acids probes.

Examples 10-17 represent the experimental set-up and results for amethod according to a second embodiment of the present invention,wherein the connected probe assembly is formed by hybridization of thefirst and second nucleic acids probes to their corresponding target DNAtemplate strands. Herein, the first nucleic acid probe of each probe setis allowed to hybridize to a first target DNA template strand, and thesecond nucleic acid probe of each probe set is allowed to hybridize toits opposite target DNA template strand.

Example 1 Design of Probes and Primers

Viral and bacterial sequences were obtained from GenBank and Los Alamosdatabase. Alignments (Clustal X v. 1.8.1) were performed on a set ofsequences for each virus to identify highly conserved regions. Sincesome of the target pathogens are RNA viruses, a reverse transcriptasestep followed by pre-amplification step was performed in order to obtaina plurality of different target DNA templates. Based upon these highlyconserved regions, reverse transcriptase (RT)-PCR primers and ligationprobes were designed. Table 1 lists the target regions, GenBankaccession numbers and position for the sequences that serve as atemplate for the hybridisation of the first and second nucleic acidprobes.

Conserved regions in the matrix protein gene (M1) were selected as thetarget for amplification and detection of influenza A virus. Theseprimers are suitable for the amplification of a variety of strains,including, but not limited to, H3N2, H2N2, H4N2, H3N8, H4N6, H6N3, H5N2,H3N6, H6N8, H5N8, H1N1, H7N1, H6N2, H9N2, H6N1, H7N3, H11N1, H5N3, H8N4,H5N1, H4N9, H4N8, H4N1, H10N8, H10N7, H11N6, H12N4, H11N9, H6N6, H2N9,H7N7, H10N5, H12N5, H11N2.

Conserved regions in the matrix protein gene (M1) were also selected asthe target for amplification and detection of influenza B virus.

Conserved regions in the major nucleocapsid protein gene (N) wereselected as target for amplification and detection of the respiratorysyncytial viruses A and B and the human metapneumovirus. Primers used toamplify human metapneumovirus and probes for their detection aresuitable for the amplification and detection of all four geneticlineages (A1, A2, B1, B2).

Conserved regions in the major outer membrane gene (OmpA) were selectedas the target for amplification and detection of Chlamydia pneumoniae.

Conserved regions in the cytadhesin P1 gene (P1) were selected as thetarget for amplification and detection of Mycoplasma pneumoniae.

Conserved regions in the macrophage infectivity potentiator gene (Mip)were selected as the target for amplification and detection ofLegionella pneumophila.

Conserved regions in the polyprotein gene (PP) were selected as thetarget for amplification and detection of encephalomyocarditis virusused as an internal control.

TABLE 1 Target genes of respiratory viruses and bacteria AccessionDisease agent Target gene Position (nt) number Influenza A virus Matrixprotein gene (M1) 194-264 CY017444 Influenza B virus Matrix protein gene(M1)  44-110 CY018438 Respiratory syncytial Major nucleocapsid proteingene (N) 1142-1221 U39661 virus A Respiratory syncytial Majornucleocapsid protein gene (N) 1352-1434 AF013254 virus B Humanmetapneumovirus Nucleocapsid protein gene (NP) 483-567 DQ843658Chlamydia pneumoniae Major outer membrane gene (OmpA) 123-201 AF347608.1Mycoplasma pneumoniae Cytadhesin P1 gene (P1)  96-171 X07191.1Legionella pneumophila Macrophage infectivity potentiator  25-103AF095223.1 gene (Mip) Encephalomyocarditis Polyprotein gene (PP)5164-5227 X00463.1 virus = internal amplification control = IAC

Table 2 lists the sequence of the RT-PCR primers (forward and reverse)used for reverse transcription and subsequent pre-amplification.

TABLE 2 RT-PCR primers of respiratory viruses and bacteria DiseaseSequence SEQ agent Primer (5′ → 3′) ID No Influenza forwardCAAGACCAATCCTG  1 A virus TCACCTCT reverse ATCGATGGCGCATG  2 CAACTGGCAAGInfluenza forward ATGTCGCTGTTTGG  3 B virus AGACACAATTG reverseGCATCTTTTGTTTT  4 TTATCCATTC Respiratory forward TCCCATAATATACA  5syncytial AGTATGATCTCAA virus A reverse AACCCAGTGAATTT  6 ATGATTAGCARespiratory forward TGTGGTATGCTATT  7 syncytial AATCACTGAAGA virus Breverse GGAGCCACTTCTCC  8 CATCTC Human forward CAAAGAGGCAAGAA  9metapneumovirus AAACAATGG reverse GCCTGGCTCTTCTG 10 ACTGTGGTCTCChlamydia forward GGAACAAAGTCTGC 11 pneumoniae GACCAT reverseAAACAATTTGCATG 12 AAGTCTGAGAA Mycoplasma forward GGTTCTTCAGGCTC 13pneumoniae AGGTCA reverse GGGGTGCGTACAAT 14 ACCATC Legionella forwardTTAGTGGGCGATTT 15 pneumophila GTTTTTG reverse ATAGCGTCTTGCAT 16 GCCTTTInternal forward ACATGTAACCGCCC 17 Amplification CCATT control (IAC)reverse TCCACGCACGCACT 18 ACTATG

The sequences of the first and second nucleic acid probes used arelisted in Table 3. As described above in Table 1 for the primers, theseprobe sets are suitable for the detection of a variety of strains. Forinstance, the influenza probes are suitable for the amplification of avariety of strains, including, but not limited to, H3N2, H2N2, H4N2,H3N8, H4N6, H6N3, H5N2, H3N6, H6N8, H5N8, H1N1, H7N1, H6N2, H9N2, H6N1,H7N3, H11N1, H5N3, H8N4, H5N1, H4N9, H4N8, H4N1, H10N8, H10N7, H11N6,H12N4, H11N9, H6N6, H2N9, H7N7, H10N5, H12N5, H11N2. The probes forhuman metapneumovirus are suitable for the detection of all four geneticlineages.

The first and second tag sequences are underlined in Table 3, whereasthe target specific regions are not. The detection sequences are initalics.

Primers used to amplify the connected probe assembly are shown in Table4. The reverse primer carries an internal label.

TABLE 4 primer 1 and primer 2 Position Sequence SEQ Primers Label label(5′ → 3′) ID No Forward — — GGGTTCCCTAAG 37 primer 1 GGTTGGA Reverse FAMinternal  GTGCCAGCAAGA 38 primer 2 label at TCCAATCTAGA position 20

TABLE 3 First and second nucleic acid probes for thedetection of respiratory viruses and bacteria. SEQ Disease agent ProbeSequence (5′ → 3′) ID No Influenza First nucleicGGGTTCCCTAAGGGTTGGACCATGCACGCTCACC 19 A virus acid probeGTGCCCAGTGAGCGAGG Second nucleic ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTC 20acid probe AATGGGAATG-ACTAGGAGAGTGGTCA-TCTAGA TTGGATCTTGCTGGCACInfluenza First nucleic GGGTTCCCTAAGGGTTGGAGACAGAAGATGGAGA 21 B virusacid probe AGGCAAAGCAGA Second nucleicACTAGCAGAAAAATTACACTGTTGGTTCGGTGGG 22 acid probeAAAGAA-ACTAGGAGAGTGGTCA-TCTAGATTGG ATCTTGCTGGCAC RespiratoryFirst nucleic GGGTTCCCTAAGGGTTGGAGGCTCTTAGCAAAGT 23 syncytial acid probeCAAGTTGAATGATACACTC virus A Second nucleicAACAAAGATCAACTTCTGTCATCCAGCAAATACA 24 acid probeCCATCCAACGGA-CATGCCTAATGGTCCAGT-TC TAGATTGGATCTTGCTGGCAC RespiratoryFirst nucleic GGGTTCCCTAAGGGTTGGAGTCCAGGTTAGGAAG 25 syncytial acid probeGGAAGACACTATAAAGATACTT virus B Second nucleicAAAGATGCTGGATATCATGTTAAAGCTAATGGAG 26 acid probeTAGATATAACAA-TCTCCACAGGTAAATCT-TCT AGATTGGATCTTGCTGGCAC HumanFirst nucleic GGGTTCCCTAAGGGTTGGAGCTCATGCATCCCAC 27 metapneumovirusacid probe AAAATCAGAGGCCTTCAGCACCAG Second nucleicACACACCAATAATTTTATTATGTGTAGGTGCCTT 28 acid probeAATATTCACTAAACTAGCATCAA-ACGGATGCAA TAGAACTCTTCGCGC-TCTAGATTGGATCTTGCTGGCAC Chlamydia First nucleic GGGTTCCCTAAGGGTTGGACCATACATTGGAGTA 29pneumoniae acid probe CAATGGTCTCGAGCAACT Second nucleicTTTGATGCTGATAACATCCGCATTGCTCAGCCAA 30 acid probeAACTACCTACAG-CAGGTCGTTACGTGGATTAGC GGTC-TCTAGATTGGATCTTGCTGGCACMycoplasma First nucleic GGGTTCCCTAAGGGTTGGAGTGGCTTGTGGGGCA 31pneumoniae acid probe GTTACCAAGCAC Second nucleicGAGTGACGGAAACACCTCCTCCACCAACAACCTC 32 acid probeGCGCCTAATACT-TCCGTCCTIAGAGTCCGCT-T CTAGATTGGATCTTGCTGGCAC LegionellaFirst nucleic GGGTTCCCTAAGGGTTGGAGCTGTTATGGGGCTT 33 pneumophilaacid probe GCAATGTCAACAGCAAT Second nucleicGGCTGCAACCGATGCCACATCATTAGCTACAGAC 34 acid probeAAGGATAAGTTGT-AGCCAGAGTGGTTTAATG-T CTAGATTGGATCTTGCTGGCAC Internal First nucleic GGGTTCCCTAAGGGTTGGAGCAGTCAGGTGAGCA 35 Amplificationacid probe CCCAGACTTGCCTCCTTGT Control (IAC) Second nucleicGAGAGGCCGCACCTTGGTAGTAAATAGACACATG 36 acid probeGCCGAGT-AGCAGCTTCTGGGCGAAGACC-TCTA GATTGGATCTTGCTGGCAC

The sequences and labels of the detection probes used are listed intable 5. Each label was positioned at the 3′ end

TABLE 5 Labelled detection probes Melting temper- ature (theo- SequenceSEQ Disease agent Label retical) (5′ → 3′) ID No Influenza ROX 55° C.TGACCACTCTCCT 39 A/B virus AGT Respiratory ROX 60° C. ACTGGACCATTAG 40syncytial GCATG virus A Respiratory Cy5 55° C. AGATTTACCTGTG 41syncytial GAGA virus B Human Cy5 70° C. GCGCGAAGAGTTC 42 metapneumovirusTATTGCATCCGT Chlamydia ROX 70° C. GACCGCTAATCCA 43 pneumoniaeCGTAACGACCTG Mycoplasma ROX 65° C. AGCGGACTCTAAG 44 pneumoniae GACGGALegionella Cy5 60° C. CATTAAGACCACT 45 pneumophila CTGGCT Internal IR70065° C. GGTCTTCGCCCAG 46 Amplification AAGCTGCT control (IAC)

Each detection probe is specific for a single pathogen as indicated,except for the detection probe for influenza virus, which detectsinfluenza A as well as influenza B.

Example 2

Sample Preparation

A nasopharyngeal lavage was used as clinical specimen. The MagnaPurenucleic acid system (Roche Diagnostics, Almere, The Netherlands) wasused as extraction method. The Total nucleic acid isolation kit and theTotal nucleic acid lysis extraction MagnaPure protocol were applied.Extractions were performed according to the manufacturer's instructions.Briefly, 200 μl of starting material was used and the purified nucleicacid was eluted in a final volume of 100 μl. Before starting theextraction, 5 μl (approximately 150 copies) of an Internal AmplificationControl (IAC) was added to the lysed sample.

Example 3 Pre-Amplification

The extracted nucleic acid with IAC was placed in a separate reactiontube and the mix of primers as shown in table 2 was added along withreagents for reverse-transcription followed by pre-amplification(RT-PCR). While any procedure known in the art for RT-PCR may be used,the following procedure was used in this example. OneStep RT-PCR(Qiagen, Hilden, Germany) was performed in 25 μl containing 5 μl OneStepRT-PCR buffer (12.5 mM MgCl2; pH 8.7 (20° C.)), 1 μl deoxy nucleosidetriphosphate (dNTP) mix (containing 1.6 μM of each dNTP), 2.5 μlprimermix (containing 2 μM of each primer), 1 μl of OneStep RT-PCREnzyme mix, 5.5 μl RNase free water and 10 μl of the extracted nucleicacid template with IAC. A blank reaction control was prepared by addingRNase free water to one reaction tube in place of nucleic acid template.The reaction tubes were placed in a Biometra T1 Thermocycler (Biometra,Goettingen, Germany) programmed as follows: 30 minutes at 50° C. reversetranscription, 15 minutes of initial PCR activation at 95° C. followedby 30 cycli of 30 seconds at 94° C., 30 seconds at 55° C. and 60 secondsat 72° C.

Example 4 Ligation and Detection of a Plurality of Different Target DNATemplates

RT-PCR reactions were 5x diluted after amplification by adding 100 μl TE(10 mM Tris-HCl, 1 mM EDTA pH 8.0) to the individual reaction tubes.Hybridisation was performed in a final volume of 8 μl consisting of 2 μlof five times diluted RT-PCR reaction, buffer components in a finalconcentration of 0.28 M KCl, 56 mM Tris-HCl pH 8.5, 0.19 mM EDTA, and acomplete mix of probes, each probe in a final concentration of 1-4 fmol.The reaction tubes were placed in a Rotor-Gene 6000 real-time system(Corbett, Sydney, Australia) programmed as follows: an initial 5 minutesdenaturation step at 98° C. followed by 1 hour at 60° C. hybridisation.Combined ligation and PCR were performed in a final volume of 40 μlconsisting of the 8 μl hybridisation reaction, buffer components in afinal concentration of 2 mM MgCl2, 3.8 mM Tris-HCl pH 8.2, 0.16 mM NAD,400 μM of each dNTP), 1 U Ligase-65, 2 U Taq-polymerase, 0.1 μM forwardprimer, 0.2 μM of an internal FAM-labelled reverse primer, eight 0.1 μM3′ end-labelled detection probes (table 5) and 0.1×SYBR® Green I(Invitrogen, Breda, The Netherlands). The following PCR conditions wereused: initial denaturation for 2 min at 95° C., followed by 40 cycles of30 seconds denaturation at 94° C., 30 seconds of annealing at 60° C. and1 minute extension at 72° C. Fluorescence was measured at the end ofeach annealing step. Excitation in each channel was at 470 nm, emissionwas detected at 510 nm, 610 nm, 660 nm and 710 nm. The addition of 0.1xSYBR Green allows the detection of an amplification curve in the 510 nmchannel independent of the label of the detection probe. Theamplification program was followed by a melting program. The meltingcurve was recorded after 2 min of denaturation at 95° C. andre-annealing at 45° C. for 90 s. Fluorescence was detected duringheating to 80° C. at 0.2° C./second and a decrease in fluorescence wasmeasured when probes melt off. Fluorescence was measured in fourchannels. Excitation in each channel was at 470 nm, emission wasdetected at 510 nm, 610 nm, 660 nm and 710 nm.

The results are presented in FIG. 7. FIG. 7 shows the melt curveanalyses of the reaction. The FAM/ROX-channel (470/610 nm) and theFAM/Cy5-channel (470/710 nm) show only background readings and nomelting peak is detected. In the FAM/Cy5-channel (470/660 nm) a meltingpeak is detected at 70° C., corresponding with the melt temperature ofthe human metapneumovirus detection probe.

Example 5 Detection of Two Different Target DNA Templates in SeparateChannels in One Reaction

In this example samples containing DNA or in vitro synthesised RNAcontaining the viral target sequences were used. Reaction 1 containedDNA of Mycoplasma pneumoniae and reaction 2 contained RNA with therespiratory syncytial virus A target sequence. Both samples alsocontained the internal amplification control. The internal amplificationcontrol was added in a concentration comparable to the concentration ofthe sample DNA or RNA. Pre-amplification and ligation and detection wereperformed as described in examples 3 and 4. The results of the melt dataof the FAM/ROX channel (470/610 nm) and the FAM/IR700 channel (470/710nm) are shown in FIG. 8. The FAM/ROX channel shows a melting peak inreaction 1 at 65° C. and in reaction 2 at 59° C. This corresponds withthe theoretical melting temperatures of the Mycoplasma pneumoniaedetection probe and the respiratory syncytial virus A detection probe.The FAM/IR700 channel shows in both samples a melting peak at 67° C.This corresponds with the theoretical melting temperature of thedetection probe of the internal amplification control.

Example 6 Quantification of Target DNA

In this example quantification of the input DNA concentration based onthe amplification curve is demonstrated. A sample containing DNA ofMycoplasma pneumoniae is 10 times diluted. Both the undiluted and thediluted sample are analyzed. Pre-amplification and ligation anddetection were performed as described in example 3 and 4. FIG. 9 showsthe amplification curve and the melt data in the FAM/ROX channel(470/610 nm) of both samples. The analysis of the melt data indicateswhether a single product is amplified in the reaction. For both samplesonly one melting peak is detected in the FAM/ROX channel. The otherchannels show only background readings. As the detected signal in theFAM/ROX channel is derived from only one product, it's valid tointerpret the amplification curve in this channel. The threshold cycle(Ct) for each sample is indicated. The difference in Ct-value betweenthe two samples is 2.2 which corresponds with a fold difference of inputDNA of 2^(2.2)=4.6.

Example 7 Detection of Two Different Target DNA Templates in One Channel

This example gives an impression of the data when two DNA targets aredetected in the same channel. The sample preparation, thepre-amplification, the ligation and detection have to be performed as inexample 2, 3 and 4. A typical result will look like the result shown inFIG. 10. Two melting peaks at 55° C. and 70° C. can be identified. Thechannel and the peak at 55° C. correspond with the ROX label and meltingtemperature of the influenza virus A and B detection probe. The secondpeak at 70° C. corresponds with the melting temperature of the ROXlabelled C. pneumoniae detection probe.

Example 8 Hybridisation, Ligation and Detection of a Plurality ofDifferent Target DNA Templates in a Single Closed Reaction Vessel

In this example an application is described which is capable ofsimultaneously detecting a plurality of different targets in clinicalsamples in a single reaction vessel. In this case a sample containingDNA of Mycoplasma pneumoniae is used. The primers and probes are thesame as described in example 1, the sample preparation andpre-amplification conditions are as described in examples 2 and 3.RT-PCR reactions were performed from the extracted nucleic acid with IACand were 5x diluted after amplification by adding 100 μl of 10 mMTris-HCl, 1 mM EDTA pH 8.0 to the individual reaction tubes. The entiremethod is performed in a single reaction vessel and hybridisation,ligation, amplification and detection is combined in one procedural stepwithout opening the reaction vessel. Reaction mixture was prepared in 20μl consisting of 2 μl of five times diluted RT-PCR reaction, buffercomponents in a final concentration of 3 mM MgCl2, 10 mM Tris-HCl pH8.2, 0.2 mM NAD, 50 mM KCl, 200 μM of each dNTP, a complete mix ofprobes, each probe in a final concentration of 1-4 fmol, 0.1 μM forwardprimer, 0.2 μM of an internal FAM-labelled reverse primer, 1 UTaq-ligase, 0.5 U HotStarTaq DNA polymerase (Qiagen, Hilden Germany),eight 3′ end-labelled detection probes (0.1 μM of each probe) (table 5)and 0.1x SYBR® Green I (Invitrogen, Breda, The Netherlands). Thereaction tubes were placed in a Rotor-Gene 6000 real-time system(Corbett, Sydney, Australia) programmed as follows: an initial 5 minutesdenaturation step at 98° C. followed by 1 hour at 60° C. hybridisation,a denaturation and initial activation of the hotstart Taq polymerase for15 min at 95° C., followed by 40 cycles of 30 s denaturation at 94° C.,30 s of annealing at 60° C. and 1 min extension at 72° C. Fluorescencewas measured at the end of each annealing step. Excitation in eachchannel was at 470 nm, emission was detected at 510 nm, 610 nm, 660 nmand 710 nm. The addition of 0.1x SYBR Green allows the detection of anamplification curve in the 510 nm channel independent of the label ofthe detection probe. The amplification program was followed by a meltingprogram. The melting curve was recorded after 2 min of denaturation at95° C. and reannealing at 45° C. for 90 s. Fluorescence was detectedduring heating to 80° C. at 0.2° C./s and a decrease in fluorescence wasmeasured when probes melt off. Fluorescence was measured in fourchannels. Excitation in each channel was at 470 nm, emission wasdetected at 510 nm, 610 nm, 660 nm and 710 nm. The result of a meltcurve analysis in the FAM/ROX channel (470/610 nm) is shown in FIG. 11.The data shows a melting peak at 64.1° C., which corresponds with themelting temperature of the Mycoplasma pneumoniae detection probe. Theother channels showed background readings.

Example 9 Clinical Validation

A total of 128 clinical specimens were analysed with a method accordingto the invention. Probe sets against influenza virus A, B and A subtypeH5N1, parainfluenza virus 1, 2, 3 and 4, respiratory syncytial virus Aand B, rhinovirus, coronavirus 229E, OC43 and NL63, adenovirus and humanmetapneumovirus were used. The sensitivity of this multiparameterrespiratory test was as good as monoplex real-time PCR for eachindividual virus. Identification of the amplified products was bymelting curve analysis using detection probes in a closed system.

Example 10 Design of Probes and Primers

Viral and bacterial sequences were obtained from GenBank and Los Alamosdatabase. Alignments (ClustalW Multiple alignment) were performed on aset of sequences for each micro-organism to identify highly conservedregions. Since some of the target pathogens are RNA viruses, a reversetranscriptase step followed by pre-amplification step was performed inorder to obtain a plurality of different target DNA templates. Basedupon these highly conserved regions, reverse transcriptase (RT)-PCRprimers and ligation probes were designed.

Table 6 lists the target regions, GenBank accession numbers and positionfor the sequences that serve as a template for the hybridisation of thefirst and second primers.

Conserved regions in the matrix protein gene (M1) were selected as thetarget for amplification and detection of influenza A virus. Theseprimers are suitable for the amplification of a variety of strains,including, but not limited to, H3N2, H2N2, H4N2, H3N8, H4N6, H6N3, H5N2,H3N6, H6N8, H5N8, H1N1, H7N1, H7N9, H6N2, H9N2, H6N1, H7N3, H11N1, H5N3,H8N4, H5N1, H4N9, H4N8, H4N1, H10N8, H10N7, H11N6, H12N4, H11N9, H6N6,H2N9, H7N7, H10N5, H12N5, H11N2, H10N8.

Conserved regions in the matrix protein gene (M1) were also selected asthe target for amplification and detection of InfB virus.

Conserved regions in the major nucleocapsid protein gene (N) wereselected as target for amplification and detection of RSV A and B andhMPV. Primers used to amplify hMPV are suitable for the amplificationand detection of all four genetic lineages (A1, A2, B1, B2).

Conserved regions in the gene encoding the Polyprotein were selected asthe target for amplification and detection of Enterovirus.

Conserved regions in the gene encoding the Polyprotein were selected asthe target for amplification and detection of Rhinovirus.

Conserved regions in the gene encoding the major outer membrane protein(OmpA) were selected as the target for amplification and detection ofChlamydophila pneumoniae.

Conserved regions in the cytadhesin protein 1 gene (P1) were selected asthe target for amplification and detection of Mycoplasma pneumoniae.

Conserved regions in the macrophage infectivity potentiator gene (Mip)were selected as the target for amplification and detection ofLegionella pneumophila.

Conserved regions in the IS481 region were selected as the target foramplification and detection of Bordetella pertussis.

A conserved region in the adjacent genes encoding the phage coat proteinand lysis protein was selected as the target for amplification anddetection of the bacteriophage MS2 used as an internal control.

Table 7 lists the sequence of the RT-PCR primers (forward and reverse)used for reverse transcription and subsequent pre-amplification.

TABLE 6 Target genes of respiratory viruses and bacteria Disease TargetPosition Accession agent gene (nt) number Respiratory Nucleoprotein1144-1202 U39661 syncytial virus A Adenovirus Hexon gene 2816-2865AB330082 Human Nucleoprotein 491-541 DQ843658 metapneumovirusRespiratory Nucleoprotein 1357-1420 AF013254 syncytial virus B InfluenzaA virus Matrix gene 117-165 CY017444 segment Influenza B virus Matrixgene  39-102 CY018438 segment L. pneumophila Macrophage  59-111AF095223.1 infectivity potentiator B.pertussis IS481 858-899 AB473880.1IRhinovirus Polyprotein gene 344-391 JN815255 Enterovirus Polyproteingene 355-426 JQ979292 C. pneumoniae Outer membrane 28-79 AF347608.1protein A M. pneumoniae Cytadhesin P1 131621-131657 CP003913.21Bacteriophage Coat protein/ 1673-1716 JF719743 MS2 lysis protein generegion

TABLE 7 (Reverse transcriptase) PCR primersused for pre-amplification of viral and bacterial nucleic acids.Sequence SEQ Disease agent Primer (5′-3′) ID No Respiratory  forwardTCCCATAATATACAA 47 syncytial GTATGATCTCAA virus A reverseAACCCAGTGAATTTA 48 TGATTAGCA Adenovirus forward GACATGACCTTCGAG 49GTGGACCCCATGGA forward GACATGACTTTTGAG 50 GTGGATCCCATGGA reverseTTATGTGGTGGCGTT 51 GCCGGC reverse TTACGTGGTAGCGTT 52 ACCGGC Humanforward CAAAGAGGCAAGAAA 53 metapneumovirus AACAATGG reverseGCCTGGCTCTTCTGA 54 CTGTGGTCTC Respiratory forward TGTGGTATGCTATTA 55syncytial ATCACTGAAGA virus B reverse GGAGCCACTTCTCCC 56 ATCTC Influenzaforward TCAGGCCCCCTCAAA 57 A virus GCC reverse GGCACGGTGAGCGTG 58 AAInfluenza forward ATGTCGCTGTTTGGA 59 B virus GACACAATTG reverseGCATCTTTTGTTTTT 60 TATCCATTC Legionella forward GGGGCTTGCAATGTC 61pneumophila AA reverse CACTCATAGCGTCTT 62 GCA Bordetella forwardGGCATCAAGCACCGC 63 pertussis TTTACCCG reverse CGGTGTTGGGAGTTC 64TGGTAGGTGTG Rhinovirus forward GCCTGCGTGGCTGCC 65 forward CCTGCGTGGCGGCC66 Enterovirus forward GCTGCGITGGCGGCC 67 Rhinovirus/ reverseGAAACACGGACACCC 68 Enterovirus AAAGTAGT Chlamydophila forwardAGAACTTAATGTGAT 69 pneumoniae CTGTAACGT reverse CGAGACCATTGTACT 70CCAATG Mycoplasma forward GCAGACGGTCGCGGA 71 pneumoniae TAACG reverseCGAACCAGGTGAGGT 72 TGCCAATG Bacteriophage forward GCAATGCAAGGTCTC 73 MS2CTAAAAGATG (Internal reverse GGAAGATCAATACAT 74 Control) AAAGAGTTGAACTTC

The sequences of the first and second nucleic acid probes are listed inTable 8. As described above in Table 7 for the primers, these probe setsare suitable for the detection of a variety of strains. For instance,the influenza probes are suitable for the amplification of a variety ofstrains, including, but not limited to, H3N2, H2N2, H4N2, H3N8, H4N6,H6N3, H5N2, H3N6, H6N8, H5N8, H1N1, H7N1, H6N2, H9N2, H6N1, H7N3, H11N1,H5N3, H8N4, H5N1, H4N9, H4N8, H4N1, H10N8, H10N7, H11N6, H12N4, H11N9,H6N6, H2N9, H7N7, H10N5, H12N5, H11N2. The probes for hMPV are suitablefor the detection of all four genetic lineages.

Primers used to amplify the primary reaction product (connected probeassembly) are shown in Table 9. The reverse primer carries an internallabel.

TABLE 8 First and second nucleic acid probes for the detection of respiratory viruses and bacteria.The first and second tag sequences areunderlined, whereas the target specific regions are not.The detection sequences are in italicsand flanked by hyphens for clear distinction. Nucleic acid SEQDisease agent probes Sequence (5′-3′) ID No Respiratory First nucleicGTGGCAGGGCGCTACGTACAAGCTCTTAGCAAAGTCAA 75 syncytical acid probeGTTGAATGATACACTC virus A Second nucleicGGACGCGCCAGCAAGATCCAATCTAGA-CATGCCTAAT 76 acid probeGGTCC-GCTGGATGACAGAAGTTGATCTTTGTT Adenovirus First nucleicGTGGCAGGGCGCTACGTACAAGTGCACAGCCICACCGC 77 acid probe First nucleicGTGGCAGGGCGCTACGTACAAGGAGTGCAICAGCCICA 78 acid probe ICGC Second nucleicGGACGCGCCAGCAAGATCCAATCTAGA-CCAATCGCAA 79 acid probeTCGTGG-CGCAGGTAIACGGIITCGATGACGCC Second nucleicGGACGCGCCAGCAAGATCCAATCTAGA-CCAATCGCAA 80 acid probeTCGTGG-CGTGCGCAGGTAIACIGIITCGATGAIGCC Human First nucleicGTGGCAGGGCGCTACGTACAAGTCAGAGGCCTTCAGCA 81 metapneumovirus acid probeCCAG Second nucleic GGACGCGCCAGCAAGATCCAATCTAGA-TAGACCAGAA 82 acid probeGTGAACGCATCT-GCACCTACACATAATAAAATTATIG GTGTGT Respiratory First nucleicGTGGCAGGGCGCTACGTACAAGGGTTAGGAAGGGAAGA 83 syncytical acid probeCACTATAAAGATACTT virus B Second nucleicGGACGCGCCAGCAAGATCCAATCTAGA-ACTAGGAGAG 84 acid probeTGGTCAGGATTGGC-CCATTAGCTTTAACATGATATCC AGCATCTTT Influenza First nucleicGTGGCAGGGCGCTACGTACAAGCTTGAGGCTCTCATGG 85 A virus acid probe AITGGCTSecond nucleic GGACGCGCCAGCAAGATCCAATCTAGA-CACGGCAGGT 86 acid probeCCGGTATCAGTTGCTTC-GAGGTGACAIGATTGGTCTT GTCTTT Influenza First nucleicGTGGCAGGGCGCTACGTACAAGTCATTIACAGAAGATG 87 B virus acid probe GAGAAGGCASecond nucleic GGACGCGCCAGCAAGATCCAATCTAGA-TAGCTCGCCA 88 acid probeTCACACGTCTCGCTGTTGACCATCTCG-ACAGTGTAAT TTTTCTGCTAGTTCTGCTT LegionellaFirst nucleic GTGGCAGGGCGCTACGTACAAGCTGCAACCGATGCCAC 89 pneumophilaacid probe ATCAT Second nucleic GGACGCGCCAGCAAGATCCAATCTAGA-GAAGTCAGTG90 acid probe TCGT-GCTATAAGACAACTTATCCTTGTCTGTAGCTA BordetellaFirst nucleic GTGGCAGGGCGCTACGTACAAGCCGAACGCTTCATCCA 91 pertussisacid probe GTCGG Second nucleic GGACGCGCCAGCAAGATCCAATCTAGA-ACTAGTAGTC92 acid probe AGGCACG-CGTAAGCCCACTCACGCAAGG Rhinovirus First nucleicGTGGCAGGGCGCTACGTACAAGCCICGTGTGCTCIITI 93 acid probe TGAITCCTCCGGFirst nucleic GTGGCAGGGCGCTACGTACAAGCGCATGTGCTTIITTG 94 acid probeTGAITCCTCCGG Second nucleic GGACGCGCCAGCAAGATCCAATCTAGA-TGTCCTTGGG 95acid probe TCCTCTTGG-AGGTTAGCCICATTCAGGGG Enterovirus First nucleicGTGGCAGGGCGCTACGAACAAGGGTGIGAAGAGICTAT 96 acid probeTGAGCTACTTIIIAITCCTCCGG Second nucleicGGACGCGCCAGCAAGATCCAATCTAGA-TGTCCTTGGG 97 acid probeTCCTCTTGG-GCTCCIIIGTTAGGATTAGCCGCATTCA GGGG Chlamydophila First nucleicGTGGCAGGGCGCTACGTACAAGGCGTAGCAACAGCTAC 98 pneumoniae acid probe TGGAACASecond nucleic GGACGCGCCAGCAAGATCCAATCTAGA-CAGGTCGTTA 99 acid probeCGTGGATTAGCGG-CATTCATGATAATTGATGGTCGCA GACTT Mycoplasma First nucleicGTGGCAGGGCGCTACGTACAAGGGCCCCIATCGCCCTC 100 pneumoniae acid probeSecond nucleic GGACGCGCCAGCAAGATCCAATCTAGA-CATTGCGTCA 101 acid probeAGTTGCTTATCAATGTTGAGCGTAAGA-TGGACCAGGG CGAAGTACGITTCAAACGG BacteriophageFirst nucleic GTGGCAGGGCGCTACGTACAAGTTTGAATGGCCGGCGT 102 MS2 acid probeCTATTAG (Internal Second nucleic GGACGCGCCAGCAAGATCCAATCTAGA-AGCAGCTTCT103 Control) acid probe GGGCGAAGACCAC-GCAGCAAACTCCGGCATCTA

TABLE 9 Primer 1 and Primer 2. Position Sequence SEQ Primers Label label(5′-3′) ID No Primer 1 — GTGGCAGGGCGCTA 104 CGAACAA Primers 2 FAMInternal GGACGCGCCAGCAA 105 label at GATCCAATCTAGA position 22

The sequences and labels of the detection probes used are listed intable 10. Each label was positioned at the 3′ end.

Each detection probe is specific for a single pathogen as indicated,except for the probe that detects both Rhinovirus and Enterovirus.

TABLE 10 Melting temper- ature 3′ (theo- Sequence SEQ Disease agentLabel retical) (5′-3′) ID No Bacteriophage BHQ1 73° C. T*CACGAGC 106 MS2TACAGCAGG (Internal T Control) Amplification BHQ1 63° C. G*TGGTCTT 107control CGCCCAGAA GCTGCT Respiratory ROX 56° C. G*GACCATT 108 syncyticalAGGCATG virus A Adenovirus ROX 61° C. C*CACGATT 109 GCGATTGG Human ROX66° C. A*GATGCGT 110 metapneumovirus TCACTTCTG GTCTA Respiratory ROX 69°C. G*CCAATCC 111 syncytical TGACCACTC virus B TCCTAGT Influenza ROX 74°C. G*AAGCAAC 112 A virus TGATACCGG ACCTGCCGT G Influenza ROX 79° C.C*GAGATGG 113 B virus TCAACAGCG AGACGTGTG ATGGCGAGC TA Legionella Cy553° C. A*CGACACT 114 pneumophila GACTTC Bordetella Cy5 58° C. C*GTGCCTG115 pertussis ACTACTAGT Rhinovirus/ Cy5 63° C. C*CAAGAGG 116 EnterovirusACCCAAGGA CA Chlamydophila Cy5 69° C. C*CGCTAAT 117 pneumoniae CCACGTAACGACCTG Mycoplasma Cy5 73° C. G*TCTTACG 118 pneumoniae CTCAACATTGATAAGCAA CTTGACGCA ATG *represents a phosphorothioate bond, which is aspecial bond between the 1^(st) and 2^(nd) nucleotide and protects theprobes against the 5′-3′ exonuclease activity of the Taq polymerase.

Example 11 Sample Preparation

Manual Sample Preparation

A nasopharyngeal swab resuspended in 1 mL of Universal Transport Medium(UTM, Copan) was used as clinical specimen. The sample input volume forthe extraction was 200 μl. All samples were spiked with 5 μL of the MS2Internal Control and extracted using QIAamp MinElute Virus SPIN Kit witha final elution volume of 60 μl according to the manufacturer'sinstructions. Negative control samples were prepared by extraction ofonly UTM, spiked also with 5 μL of the MS2 internal control.

Automated Sample Preparation

A nasopharyngeal swab resuspended in 1 mL of Universal Transport Medium(UTM, Copan) was used as a clinical specimen. The sample input volumefor the extraction was 200 μl. Automated nucleic acid extraction withmagnetic silica based on the protocol according to Boom (Boom 1990) wasperformed using the easyMAG (bioMérieux), according to the ‘Generic’protocol in the manufacturer's instructions. Lysis was performedon-board and the elution volume was 60 μl. The Internal Control (5.5 μLper sample) was added together with the silica.

Example 12 Pre-Amplification

While any procedure known in the art for RT-PCR may be used, thefollowing procedure was used in this example. One-step RT-PCR wasperformed using 1x Fast Virus 1-step master mix from Life Technologies,200 nM of each of the pre-amplification primers as shown in Table 7, and10 μL of the extracted nucleic acid.

The reaction tubes were placed in a Biometra T1 Thermocycler (Biometra,Goettingen, Germany) programmed as follows: 10 minutes at 50° C. reversetranscription, 2 minutes of initial PCR activation at 95° C. followed by40 cycli of 20 seconds at 94° C., 20 seconds at 55° C. and 35 seconds at72° C.

Example 13 Hybridisation, Formation of Connected Probe Assembly andDetection of a Plurality of Different Target DNA Templates in a SingleClosed Reaction Vessel

Combined formation and amplification of the connected probe assembly andthe detection of the amplified connected probe assembly were performedin a final volume of 25 μl consisting of 5 μl of the pre-amplificationreaction product, buffer components in a final concentration of 1.5 mMMgCl2, 10 mM Tris-HCl pH 8.5, 50 mM KCl, 200 μM of each dNTP, 1.25 UTaq-polymerase, 80 nM of each first nucleic acid probe (Table 8), 20 nMof each second nucleic acid probe (Table 8), 50 nM of primer 1 (Table9), 400 nM of the internally FAM-labelled primer 2 (Table 9), andthirteen 200 nM 3′ end-labelled detection probes (table 10) (IntegratedDNA Technologies). The following PCR conditions were used: initialdenaturation for 2 min at 95° C., followed by (i) 10 cycles of 15seconds denaturation at 94° C., 15 seconds of annealing at 55° C. and 15second extension at 72° C. and (ii) 23 cycles of 15 seconds denaturationat 94° C., 15 seconds of annealing at 50° C. and 15 second extension at72° C. Fluorescence was measured at the end of each annealing step.Excitation in each channel was at 470 nm, emission was detected at 510nm, 610 nm and 660 nm. The amplification program was followed by amelting program. The melting curve was recorded after 2 min ofdenaturation at 95° C. and re-annealing at 40° C. for 90 s. Fluorescencewas detected during heating to 90° C. at 1° C./second and a decrease influorescence was measured when probes melt off. Fluorescence wasmeasured in three channels. Excitation in each channel was at 470 nm,emission was detected at 510 nm, 610 nm and 660 nm.

Example 14 Detection of RNA from Two Different Respiratory Viruses inSeparate Channels in One Reaction

In this example, a clinical specimen is shown that contained bothInfluenza A and Enterovirus/Rhinovirus. The sample preparation, thepre-amplification, the ligation and detection were performed as inexample 2, 4 and 5. Fluorescence was measured in three channels.Excitation in each channel was at 470 nm, emission was detected at 510nm (for FAM), 610 nm (for ROX) and 660 nm (for Cy5). The results of themelt data are shown in FIGS. 13A-C. The ROX channel shows a melting peakat 75° C. corresponding with the theoretical melting temperature of thedetection probe of Influenza A. The Cy5 channel shows a melting peak at65° C. corresponding with the theoretical melting temperature of thedetection probe of Enterovirus/Rhinovirus. The FAM channel shows anegative melting peak at 73° C. corresponding with the theoreticalmelting temperature of the detection probe of the Internal Control, anda negative melting peak at 63° C. corresponding with the Amplificationcontrol of mix 1.

Example 15 Detection of Two Different Target DNA Templates in OneChannel

This example gives an impression of the data when two DNA targets aredetected in the same channel. The sample preparation, thepre-amplification, the ligation and detection have to be performed as inexample 2, 3 and 4. A typical result will look like the result shown inFIG. 14. Two melting peaks at 61° C. and 75° C. can be identified in theROX channel. The peak at 61° C. corresponds with the melting temperatureof the Adenovirus detection probe. The second peak at 75° C. correspondswith the melting temperature of the Influenza A detection probe.

Example 16 Appearance of the Amplification Controls in Two DifferentReaction Mixes

This example (FIGS. 15A-B) gives an impression of the data when a sample(in this case a sample negative for any pathogen detected in the assay)is tested in two mixes, of which each mix contains a recognizableAmplification Control that allows distinction of mix 1 and 2 bydifferences in melting temperature. The negative result of the sample isvalidated by the presence of the Internal Control in both mixes and bythe presence of the respective Amplification Controls.

Example 17 Clinical validation

A total of 195 nasopharyngeal swabs from the consortium for “Genomics tocombat resistance against antibiotics for community-acquired lowerrespiratory tract infection (LRTI) in Europe (GRACE)” (Finch 2012) wereanalysed according to Examples 2, 4 and 5. These samples were tested inan embodiment of the invention that allows detection of 22 respiratorypathogens. After a multiplex pre-amplification step, the reaction wasperformed in two mixes. Mix 1 contained the assembly probes anddetection probes for Adenovirus, Metapneumovirus, Influenza virus A andB, Respiratory syncytial virus A and B, Rhinovirus/Enterovirus (notdistinguished) Bordetella pertussis, Legionella pneumophila, Mycoplasmapneumoniae and Chlamydophila pneumoniae. Mix 2 contained the assemblyprobes and detection probes for A(H1N1)pdm09, Parainfluenza virus1-2-3-4, Bocavirus, Coronavirus NL63/HKU1 (not distinguished),Coronavirus OC43 and Coronavirus 229E. The overall (average) sensitivityof this embodiment of the invention was 91.1% and the specificity 99.6%.

What is claimed is:
 1. A method for detecting and differentiating aplurality of pathogenic organisms having a copy number of less than 6000in a clinical sample in a single reaction vessel, the clinical samplecomprising a plurality of different target RNA templates and/or aplurality of different target DNA templates derived from the pluralityof pathogenic organisms, each target DNA template comprising a firsttarget segment and a second target segment, the combination of both thefirst target segment and the second target segment being specific for aparticular target DNA template, wherein the first target segment and thesecond target segment are essentially adjacent to one another andwherein the first target segment is located 3′ from the second targetsegment, said method comprising the steps of: (a) a reversetranscription step in the single reaction vessel of the plurality ofdifferent target RNA templates into the plurality of different targetDNA templates and/or an optional pre-amplification step of the pluralityof different target DNA templates in the single reaction vessel; (b)bringing said plurality of different target DNA templates into contactwith a plurality of different probe sets in the single reaction vessel,each probe set being specific for a particular target DNA template ofthe plurality of different target DNA templates and allowing theparticular target DNA template to hybridise thereto, each probe setcomprising: a first nucleic acid probe having a first target regionhybridisable to the first target segment of said particular target DNAtemplate and a first tag region, wherein the first tag region is located5′ from the first target region and comprises a first tag sequence, anda second nucleic acid probe having a second target region hybridisableto the second target segment of said particular target DNA template anda second tag region, wherein the second tag region is located 3′ fromthe second target region and comprises a second tag sequence, andwherein at least one of the first nucleic acid probe or the secondnucleic acid probe contains a detection sequence located 5′ from thesecond tag sequence or located 3′ from the first tag sequence; (c)forming a plurality of connected probe assemblies in the single reactionvessel, wherein the plurality of connected probe assemblies comprisesthe plurality of different probe sets and is formed by hybridization ofsaid first nucleic acid probe of each probe set with its correspondingtarget DNA template strand; and hybridization of said second nucleicacid probe of each probe set with its corresponding opposite target DNAtemplate strand; (d) amplifying the plurality of connected probeassemblies in the single reaction vessel to obtain a plurality ofamplicons, wherein the plurality of connected probe assemblies areamplified by allowing the plurality of connected probe assemblies tocontact with a plurality of nucleic acid primer pairs, each nucleic acidprimer pair comprising: a primer 1 and a primer 2, wherein at least oneof primer 1 or primer 2 comprises at least one internal donor oracceptor fluorescent label at or near its 3′ end, thereby providing aplurality of internally labelled amplicons upon amplification of saidplurality of connected probe assemblies, wherein the internal donor oracceptor fluorescent label is incorporated in the first or second tagregion and is essentially adjacent to the detection sequence; and (e)detecting and differentiating the plurality of internally labelledamplicons in the single reaction vessel, wherein the plurality ofinternally labelled amplicons are detected and differentiated byperforming a real-time melting curve analysis comprising: (1) providinga plurality of labelled detection probes, each labelled detection probebeing specific for a particular detection sequence of a particularinternally labelled amplicon and comprising: at least one fluorescentdonor or acceptor label complementary to the internal donor or acceptorfluorescent label incorporated in the first or second tag region by saidplurality of nucleic acid primer pairs, wherein a single pair of donorand acceptor labels is used in detecting a plurality of internallylabelled amplicons, wherein each similarly labelled detection probeexhibits a different melting temperature upon hybridisation to theparticular detection sequence of the particular internally labelledamplicon, such that the plurality of internally labelled amplicons isdistinguishable; and a nucleic acid region specifically hybridisable tosaid particular detection sequence of the particular internally labelledamplicon, (2) allowing the plurality of internally labelled amplicons tohybridise with the plurality of labelled detection probes, and (3)monitoring hybridisation and the different melting temperatures of theplurality of labelled detection probes by measuring the fluorescence ofthe acceptor labels over a pre-selected increasing temperature rangewithin different fluorescent detection channels, wherein: saidhybridisation of the plurality of labelled detection probes isindicative for the presence of the plurality of different pathogenicorganisms in the clinical sample, the different melting temperatures incombination with the different fluorescent labels of the plurality oflabelled detection probes allow detection and distinguishability of aplurality of different pathogenic organisms in the clinical sample, suchthat up to about 30 different pathogenic organisms in the clinicalsample may be detected and distinguished; and steps (a)-(e) areperformed in a closed system.
 2. The method according to claim 1 whereinstep (d) is performed in order to obtain a plurality of ampliconscomprising: the first tag region or part thereof, the first targetregion, the second target region, the detection region, and the secondtag region or part thereof, or the complements thereof.
 3. The methodaccording to claim 1 wherein the plurality of different target DNAtemplates is obtained by amplifying a plurality of different nucleicacids in the clinical sample.
 4. The method according to claim 1 whereinthe plurality of different target DNA templates is extracted from theclinical sample before allowing the plurality of different target DNAtemplates to hybridise with said plurality of different probe sets. 5.The method according to claim 1 wherein at least one of the first andsecond tag sequences have a nucleic acid sequence chosen in such a waythat the first and second tag sequences do not hybridise to theplurality of different target DNA templates.
 6. The method according toclaim 1 wherein at least one of the first and second tag regions have anucleic acid sequence chosen in such a way that the first and second tagregions do not hybridise to the plurality of different target DNAtemplates.
 7. The method according to claim 1 wherein at least one ofthe first and second tag sequences are universal sequences.
 8. Themethod according to claim 1 wherein at least one of primer 1 or primer 2is a DNA primer.
 9. The method according to claim 1 wherein thedetection sequence is immediately adjacent to the second tag sequence.10. The method according to claim 1 wherein primer 2 comprises theinternal donor or acceptor fluorescent label at or near its 3′ end. 11.The method according to claim 10 wherein the detection of the pluralityof internally labelled amplicons comprises the step of exciting thedonor fluorescent label and measuring the fluorescence of the acceptorfluorescent label.
 12. The method according to claim 1 wherein saidplurality of labelled detection probes is chosen in such a way that theindividual detection probes can be distinguished from each other bytheir difference in length or nucleotide composition.
 13. The methodaccording to claim 1 wherein the pre-selected increasing temperaturerange consists of a temperature range defined by the different meltingtemperatures of the plurality of labelled detection probes being atleast 3 degrees Celsius apart and wherein the melting curve analysis isperformed to detect a decrease in fluorescence when a labelled detectionprobe de-hybridises.
 14. The method according to claim 1 whereinhybridisation in step (b) is performed in the presence of Mg ions and inthe presence of less than 200 mM KCl.
 15. The method according to claim1 wherein a hot-start enzyme is used for amplification in step (d). 16.Kit for performing the method according to claim 1 comprising: theplurality of different probe sets, a DNA ligase, a DNA polymerase, theplurality of nucleic acid primer pairs, the plurality of labelleddetection probes comprising at least one donor or acceptor fluorescentlabel, and instructions for performing the method.
 17. The kit accordingto claim 16 wherein the plurality of labelled detection probes havedifferent melting temperatures.
 18. The kit according to claim 17wherein the melting temperatures of the plurality of labelled detectionprobes differ by at least 3 degrees Celsius.